CN103443292A - QTLs associated with and methods for identifying whole plant field resistance to sclerotinia - Google Patents

QTLs associated with and methods for identifying whole plant field resistance to sclerotinia Download PDF

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CN103443292A
CN103443292A CN201180068000XA CN201180068000A CN103443292A CN 103443292 A CN103443292 A CN 103443292A CN 201180068000X A CN201180068000X A CN 201180068000XA CN 201180068000 A CN201180068000 A CN 201180068000A CN 103443292 A CN103443292 A CN 103443292A
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I.法拉克
V.普里莫莫
L.图斯伊拉姆
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Abstract

Markers associated with Sclerotinia whole plant field resistance are provided. Methods of identifying Sclerotinia resistant and susceptible plants, using the markers are provided. Methods for identifying and isolating QTLs are a feature of the invention, as are QTLs associated with Sclerotinia whole plant field resistance.

Description

With whole plant the field resistance QTL be associated and the method for identifying the whole plant field resistance of Sclerotinia to Sclerotinia
the cross reference of related application
Require the U.S. Provisional Application 61 that is filed on December 22nd, 2010 to the applying date according to 35 U.S.C. § 1.19 (e), 426,170, be filed in the U.S. Provisional Application 61/449 on March 7th, 2011,776 and be filed in the U.S. Provisional Application 61/566 on December 2nd, 2011,064 rights and interests, the full text of each above-mentioned patent application is incorporated herein by reference.
Technical field
The present invention relates generally to molecular biology of plants.More specifically, it relates in Btassica the quantitative trait locus (QTL) be associated with whole plant field resistance to Sclerotinia, and identifies in Btassica and other plant species the whole plant field resistance to Sclerotinia with these QTL.
Background technology
Sclerotinia (Sclerotinia) infects the 400 kind of plant species that surpass of Canada whole area, comprise numerous important crops economically, for example Btassica (Brassica) species, Sunflower Receptacle, dry beans, field pea, French beans and potato (Boland and Hall (1994) can.J.Plant Pathol.16:93-108).Sclerotinite (Sclerotinia sclerotiorum) is the reason that surpasses this disease of 99%, and sclerotinia species (Sclerotinia minor) causes being less than this disease of 1%.Sclerotinia produces sclerotium, and sclerotium is the erose dark color that can survive in soil four to the five years body of surviving the winter.Depend on envrionment conditions and crop canopies, sclerotium can produce sporophore ground or produce mycelia ground and germinate.These two kinds different germination types cause two kinds of different Damage Types.Produce the sclerotium germinateed on sporophore ground and produce infection apothecium and the thecaspore of tissue on the ground, cause cane blight, stem rot, a maize ear rot, pod rot, southern blight and the blossom blight of plant.Produce the sclerotium germinateed on mycelia ground and produce the mycelia that can infect root tissue, cause crown rot, root rot and base rot disease.
Sclerotinia comprises during draw Kano and causes the sclerotium stem rot in Btassica, also is southern blight.Draw Kano is a kind of Brassica plants that has low-level glucosinolates and erucic acid in seed.Sclerotium produces sporophore ground in summer and germinates, and produces apothecium.Apothecium discharges the wind-borne thecaspore that is transmitted to many kms.Before flower is pulled open in Kano and during, this disease is subject to the temperature promotion of damp soil condition (at least 10 days or approaching field capacity) and 15-25 ℃.Spore can not direct infection leaf and stem.They must first drop on the petal and the pollen on stem and leaf of spending, falling.Petal age effects efficiency of infection, older petal can infect (people (1999) " The Effect of Petal Characteristics, Inoculum Density and Environmental Factors on Infection of Oilseed Rape by Sclerotinia sclerotiorum " the The Regional Institute Ltd. such as Heran more http:// www.regional.org.au/au/gcirc/3/428.htm).The part of fungal spore utilization flower is germinateed and infection plant as food source.
Root rot can also occur in Btassica under certain conditions.For example, winter habit and spring habit Kano draw occasional at Europe (draw the winter habit Kano) and Georgia, and root rot occurs the winter of the gentleness of US (draw the spring habit Kano).
The severity of Sclerotinia in Btassica is variable, and depends on time of infection and weather condition (people such as Heran, referring to above).This disease is promoted by low temperature and long precipitation.Temperature between needing 20 and 25 ℃ of optimal plant infection and be greater than 80% relative humidity (people such as Heran, referring to above).For indivedual fields, reported the loss (Manitoba Agriculture, Food and Rural Initiatives, 2004) of from 5 to 100% scopes.Fifty-fifty, production loss equals 0.4 to 0.5 and is multiplied by infection per-cent.For example, if field has 20% infection (20/100 plant infected), production loss will be approximately 10% so.In addition, Sclerotinia can also cause heavy losses in wet harvested material.
The symptom that Sclerotinia infects occurs usually in several weeks after the beginning of blooming.Above soil line place or its and on top branch and beanpod, plant occurs light grey to white pathology.Infection occurs through be everlasting leaf and stem joint, because infected petal is embedded in there.The stem infected show as bleach and tend to tear.Hard black fungi sclerotium is grown in the stem, branch or the beanpod that infect.The plant infected when blooming produce seldom or do not produce seed.Plant with stem of winding withers with ripe prematurely.The crop of severe infections often lodges, damages when windrow and make windrow more consuming time.Infection can occur at whole ground plant parts, especially in dense or district's sheet lodging.Once plant is infected, mould grows and invades healthy tissue constantly in stem.New sclerotium will form, disease is extended to next season.
Kano with some Morphologic Characters draws kind more can resist the Sclerotinia infection.For example, the kind (turnip (Brassica rapa)) of Poland has lighter canopy, and seems to have much lower infection level.In addition, do not have petalled kind (without the petal kind) the initial source of infection (that is, petal) is not provided and has avoided the Sclerotinia infection (people (1995) such as Okuyama with larger degree bulletin of the Tohoku National Agricultural Experiment Station.National Agriculture Research Center, Tsukuba, Ibaraki 305, JAP 3-1-1an.89:11-20; Fu (1990) acta Agriculture Shanghai.economic Crop Research Institute, Jiangsu Province Academy of Agricultural Sciences, Nanjing210024, China 6 (3): 76-77.The example of giving the Morphologic Characters of the susceptibility that Sclerotinia is to a certain degree reduced in Btassica comprises the lodging resistance of raising, lower petal reservation, higher branch (degree and position), bloom (early starting and/or short lasting) and leaf abscission early.Jurke and Fernando (" Plant Morphology of Canola and its Effects on Sclerotinia sclerotiorum infection in ICPP " 2,003 8 thinternational Congress of Plant Pathology, New Zealand) having screened 11 kinds of Kanos for Sclerotinia disease incidence draws genotype.Explained the noticeable change of disease incidence by plant morphology, and the difference that petal retains is accredited as most important factor.Yet these Morphologic Characters are not given separately the resistance to Sclerotinia, and to draw strain to be considered to Sclerotinia be susceptible in Canadian most of Kano.
The Main Means of controlling Sclerotinia in infected farm crop is to spray mycocide.Comprise the Rovral from Bayer for controlling the typical mycocide of Sclerotinia on Btassica tM/ Proline and from the Ronilan of BASF tM/ Lance tM.If infect, be that significantly applications of fungicides is otiose, because too late and can't onset.Therefore, the grower must be assessed their field for pest and disease risk, whether to determine applications of fungicides.This can be by the look-up table that provides with government or by being undertaken by the petal test kit.Which kind of mode no matter, the method for using is all very loaded down with trivial details and be easy to make mistakes.
Carried out making great efforts in a large number to cultivate the spring habit Brassica plants of Sclerotinia resistance.With control Sclerotinia by applications of fungicides and compare, inherent resistance will be convenient, economical and environment amenable.Because this proterties is polygenic, it will be stable, and can as mycocide, effect easily not change.It is also susceptible that the winter habit Kano is drawn Sclerotinia.
The spring habit Kano draws (swede type rape mutation (Brassica napus subsp.oleifera var.annua)) and winter habit Kano to draw the difference of (swede type rape mutation (Brassica napus subsp.oleifera var.biennis)) mainly to be not need to force vernalization treatment.The Semen Brassicae campestris in Asia and Kano draw kind to have low extremely medium demand for vernalization treatment.Although draw when when being planted spring, completing its reproductive cycle the winter habit Kano, if the kind in Asia was planted and can not be completed its reproductive cycle in late spring, plant when early spring and suffer cold to make the kind in Asia to bloom and to set seeds.Under controlled condition, winter variety needs the vernalization treatment in 12 to 14 week, and the kind in Asia needed for 2 to 8 week.Table 1 has been summed up winter habit, semi-winterness (Asia) and spring habit Kano and has been drawn the difference between kind.
table 1: the main mensuration of the habit of swede type rape (Brassica napus) material
Figure BDA00003691347400041
*with regard to draw the spring habit Kano, Canada, Europe and Australian spring varieties can be planted and be grown at any environment and sowing time.
Some Chinese Cultivars that draw Semen Brassicae campestris/Kano are partial resistances to Sclerotinia.For example, the people ((2003) such as ChunYun acta Agronomica Sinica29 (5): 715-718); The people such as HanZhong ((2004) scientia Agricultura Sinica37 (1): 23-28); The people such as WeiXin ((2002) chinese Journal of Oil Crop Sciences24 (3): 47-49); The people such as YongJu ((2000) chinese Journal of Oil Crop Sciences22 (4): the kind of 1-5) having described the partial resistance of Semen Brassicae campestris.Yet some in these kinds are not that quality is drawn in Kano, and they all need vernalization treatment.The part field resistance of the kind of China originates from Semen Brassicae campestris kind Zhong you 821.Although Zhong you 821 has the improvement of partial resistance, its reaction to pathogenic agent under the envrionment conditions of the growth that is conducive to Sclerotinia is more unsettled (people (1999) " Breeding; inheritance; and biochemical studies on Brassica napus cv.Zhongyou 821:Tolerance to Sclerotinia sclerotiorum (stem rot) " the .Proceedings of the 10th International Rapeseed Congress such as Li, Canberra, Australia).
The Japanese Cultivar of some of Semen Brassicae campestris has the part stem resistance to Sclerotinia.Part stem resistance be with the winter habit Kano draw relatively in the (people (1987) " A field study of rapeseed (Brassica napus) resistance to Sclerotinia sclerotiorum. " 7 such as Brun that detects by indoor test thinternational Rapeseed Congress, Poznan, Poland).Yet, these kinds be not Kano draw quality and be semi-winterness type (referring to table 1).
Due to the quantity character of this proterties, drawing middle cultivation is very difficult in Kano to the resistance of Sclerotinia.In addition, the physiology resistance has doubled for the complexity of resistance breeding with the integration of avoiding or reduce the Morphologic Characters of infection.In addition, because the direct environment and heredity (GXE) with plant interacts (that is, temperature and humidity requirement, and microenvironment requirement), antagonistic screening is very difficult.As described above, although have coevolution and environmental stress for many years to be selected for this proterties, only have Canadian spring habit Btassica kind seldom to there is the resistance to Sclerotinia.In China, obtained the field resistance of (and recently in kind is drawn in some Kanos) certain level in the Semen Brassicae campestris by breeding work, as the description to Zhong you 821 (people such as Li, referring to above).Yet, the level of this type of partial resistance or tolerance is relatively low, and it is still (world-of-mouth communication) recommended (people (1999) " Effect of cultural control on rapeseed stem rot (Sclerotinia sclerotiorum) in Brassica napus. " the Proceedings of the 10th International Rapeseed Congress such as Hu that the using of mycocide drawn kind for the whole Semen Brassicae campestris in China and Kano, Canberra, Australia).Other breeding work comprises quantitative trait locus analysis (Zhao and Meng (2003) for instance theoretical and Applied Genetics106 (4): 759-764), the selection by mutation (people (1999) such as Mullins european Journal of Plant Pathology105 (5): 465-475; The people such as Wu (1996) sichuan Daxue Xuebao (Ziran Kexueban)33 (2): 201-205; The people such as LiangHong, 2003, a large amount of screening operations (people (1989) such as Sedun canadian Journal of Plant Science69 (1): 229-232; The people such as Zhao (2004) plant Disease88 (9): 1033-1039); With the screening (people (2004) such as Li to expressed sequence tag (EST) fungal Genetics and Biology41 (8): 735-753).Cultivate several spring habits Kano that Sclerotinia is had to a medium tolerance and drawn the kind (people (2000) such as Ahmadi seed and Plant16 (1): Pe127-Pe129, en14; The people such as Ahmadi (2000) Introduction of rapeseed (Brassica napus L.), cultivar Esteghlal.Seed and Plant 16 (1): Pe127-Pe126, en13; The people such as BaoMing (1999) chinese Journal of Oil Crop Sciences4:12-14; With people (1991) such as Liu scientia agricultura Sinica24 (3): 43-49), yet the level of tolerance is low, and these kinds can not tolerate high disease pressure.Recently, cultivate the transgenosis Kano of carrying oxalate oxidase gene and drawn (United States Patent (USP) 6,166,291 and division patent), yet had supervision and the social concern relevant to transgenic plant.Kind is drawn in the Kano that needs significant technical manual intervention to cultivate anti-Sclerotinia.
More nearest, after long-term and the high-intensity procedure of breeding, cultivated to Sclerotinia have the Btassica of high-caliber resistance and Kano draw kind (referring to, for example, WO 2006/135717, it all instructs content to be incorporated herein by reference).The method is time and work highly dense type, and needs the long time to determine that whether the procedure of breeding is successful.For the difficulty in the breeding of Sclerotinia whole plant field resistance, be (at least in part) polygene character due to this proterties.
Needed on this area and industry is the method for utilizing molecular marked compound to identify to give to the gene of the whole plant field resistance of Sclerotinia.Then, these markers can be used to the favourable allelotrope of these genes of mark in segregating population, and are used to subsequently make the antagonistic selection of pin more effective.The invention provides above-mentioned and other advantage.
Summary of the invention
The invention provides in plant identification method and the mark of the quantitative trait locus (" QTL ") be associated with the whole plant field resistance of whole plant field resistance to Sclerotinia or raising.
The feature of first aspect of the present invention is to identify to show the Brassica plants of the whole plant field resistance of the whole plant field resistance of Sclerotinia or raising or the method for germplasm.Described method is included at least one allelotrope of at least one quantitative trait locus (QTL) that in described plant or germplasm, detection is associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising, wherein said QTL is positioned to and is selected from N1, N3, N4, N7, N8, N9, N10, N11, N12, N13, N15, the linkage group of N18 or N19, wherein each linkage group comprises at least one mark that the statistical significance with p≤0.01 is associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising, thereby evaluation will show Brassica plants or germplasm to the whole plant field resistance of the whole plant field resistance of Sclerotinia or raising.
In one embodiment, described QTL is positioned to and is selected between following chromosomal region: (a) side joint comprise (i) mark CA0614 and PE0177 or (ii) interval of mark AG0093 and AG0482 on linkage group N1; (b) side joint the interval that comprises mark CA0410 and AG0023 on linkage group N3; (c) side joint the interval that comprises mark BG1442 and BG0106 on linkage group N4; (d) side joint the interval that comprises mark AG0510 and CA0105 on linkage group N7; (e) side joint the interval that comprises mark CA0837 and BG1286 on linkage group N8; (f) side joint comprise (i) mark CA1034 and AG0441 or (ii) interval of mark AG0378 and KK66 on linkage group N9; (g) side joint the interval that comprises mark BG0228 and PE0131 on linkage group N10; (h) side joint comprise (i) mark CA0120 and CA0163 or (ii) interval of mark CA0120 and CA1097 on linkage group N11; (i) side joint comprise (i) mark BG1321 and CA0991 or (ii) interval of mark CA0753 and PE0250 on linkage group N12; (j) side joint the interval that comprises mark CA0603 and CA0736 on linkage group N13; (k) side joint the interval that comprises mark PE0286 and AG0369 on linkage group N15; (l) side joint comprise (i) mark BG0278 and CA0636 or (ii) interval of mark UB0315 and CA0739 on linkage group N18; (m) side joint comprise (i) mark CA1107 and CA0221 or (ii) interval of mark UB0307 and KK98G on linkage group N19.
In another embodiment, described QTL is positioned to and is selected between following chromosomal region: (a) the one or more intervals on linkage group N1, and its side joint also comprises mark (i) AG0093 and PE0203 or (ii) BG0111 and BG1392 or (iii) BG1090 and AG0482 or (iv) BG1090 and PE0203 or (v) CA0614 and BG1392 or (vi) BG0988 and AG0482 or (vii) AG0243 and AG0482 or (viii) AG0243 and BG1453 or BG0988; (b) the one or more intervals on linkage group N3, its side joint also comprises mark (i) BG1197 and AG0023 or (ii) CA0410 and BG1368 or (iii) CA0410 and BG1197; (c) the one or more intervals on linkage group N4, its side joint also comprises mark (i) BG1442 and BG0106 or (ii) UB0181 and BG0106; (d) the one or more intervals on linkage group N8, its side joint also comprises mark (i) BG1449 and BG1062 or (ii) CA0837 and AG0328 or (iii) CA0837 and BG1062 or (iv) CA0837 and BG1101 or (v) CA0837 and BG1286 or (vi) CA0837 and BG1449 or (vii) PE0281 and BG0647; (e) the one or more intervals on linkage group N9, its side joint also comprises mark (i) AG0323 and BG0295 or (ii) CA1034 and AG0378 or (iii) BG1123 and AG0441; (f) the one or more intervals on linkage group N10, its side joint also comprises mark (i) BG0228 and AG0047 or BG0255 and PE0131; (g) the one or more intervals on linkage group N11, its side joint also comprises mark (i) BG0031 and BG1149 or (ii) BG0031 and BG1230 or (iii) BG0031 and BG1513 or (iv) CA0120 and CA0328 or (v) PE0283 and CA0163 or (vi) PE0324 and PE0283 or (vii) CA0328 and PE0324 or (viii) CA0226 and BG0713 or (ix) CA0233 and CA1080 or (x) CA0233 and AG0370; (h) the one or more intervals on linkage group N12, its side joint also comprises mark (i) BG1321 and CA0991 or (ii) BG1321 and CA1027 or (iii) BG1321 and PE0133 or (iv) PE0063 and CA0991 or (v) PE0133 and CA0991 or (vi) CA1027 and PE0063 or (vii) CA1027 and UB0331 or (viii) CA0423 and PE0250 or (ix) AG0359 and PE0250 or (x) AG0359 and CA0896; (i) the one or more intervals on linkage group N13, its side joint also comprises mark (i) BG0516 and AG0148 or (ii) CA0488 and AG0148 or (iii) CA0488 and CA0736 or (iv) CA0603 and AG0504 or (v) BG1288 and AG0504; (j) the one or more intervals on linkage group N15, its side joint also comprises mark (i) CA0719 and AG0369 or (ii) PE0091 and PE0187 or (iii) PE0286 and AG0369 or (iv) PE0286 and PE0187 or (v) PE0286 and CA0719; (k) the one or more intervals on linkage group N18, its side joint also comprises mark (i) AG0285 and CA0636 or (ii) BG0278 and CA07739 or (iii) CA0739 and CA0636 or (iv) UB0315 and CA0636 or (v) UB0315 and CA0739; (l) the one or more intervals on linkage group N19, its side joint also comprises mark (i) CA0552 and CA0221 or (ii) CA1107 and CA0552 or (iii) CA1107 and CA0221 or (iv) CA0221 and KK98G or (v) UB0307 and BG1241 or (vi) BG1241 and KK98G or (vii) CA0221 and BG1241.
In a specific embodiment, described QTL is positioned between the chromosomal region on linkage group N1, N9, N11, N12, N18 or N19.
In other embodiments, described mark Including Polymorphism, described polymorphism is by least one Identity of allele of described at least one quantitative trait locus (QTL) for to be associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising, and described detection comprises and identifies described polymorphism.Polymorphism can be, for example, and single nucleotide polymorphism (SNP) or simple repeated sequence (SSR).In another embodiment of method of the present invention, described detection comprises at least one mark that detection comprises described polymorphism, and described mark is selected from: AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; CA1107; PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; And KK98G.
In another embodiment of method of the present invention, described detection comprises the described polymorphism detected at least one mark, and described mark is selected from: AG0093; AG0304; AG0378; AG0391; AG0482; BG1149; BG1230; BG1241; BG1453; BG1513; CA0120; CA0221; CA0546; CA0739; CA1027; PE0063; PE0203; UB0163; And UB0315.
In other embodiments, described method comprises that detection is positioned at two or more marks of two or more different linkage groups, be positioned at three or more marks of three or more different linkage groups, be positioned at four or more mark of four or more different linkage groups, be positioned at five or more mark of five or more different linkage groups, be positioned at six or more mark of six or more different linkage groups, be positioned at seven or more mark of seven or more different linkage groups, be positioned at eight or more mark of eight or more different linkage groups, be positioned at nine or more mark of nine or more different linkage groups, be positioned at ten or more mark of ten or more different linkage groups, be positioned at ten one or more marks of ten one or more different linkage groups, or be positioned at 12 or more mark of 12 or more different linkage groups.
In other embodiments, in described method, described detection comprises from the genomic dna of described plant or the germplasm described mark that increases, and determines the described polymorphism be associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising that marks whether to comprise.In other embodiments, described plant is swede type rape (Brassica napus); Leaf mustard (Brassica juncea); Turnip (Brassica rapa); Wild cabbage (Brassica oleracea); Or brassicacarinata (Brassica carinata).In other embodiments, described plant is that draw the spring habit Kano, draw the winter habit Kano or draw the semi-winterness Kano.In another embodiment, the whole plant field resistance of described whole plant field resistance or raising is to compare the disease incidence of reduction due to the allelic plant with lacking described QTL, and the allelotrope of described QTL is associated with the whole plant field resistance of described whole plant field resistance or raising.In another embodiment, the whole plant field resistance of described whole plant field resistance or raising is to compare the disease severity of reduction due to the allelic plant with lacking described QTL, and the allelotrope of described QTL is associated with the whole plant field resistance of described whole plant field resistance or raising.In another embodiment, described plant has the whole plant field resistance of sclerotinite or the whole plant field resistance of raising.
The feature of another aspect of the present invention is that plant by identifying according to method mentioned above or filial generation and the second plant cross-pollination make the Sclerotinia resistance infiltrate the method in the second plant, and wherein said the second plant lacks at least one allelotrope of at least one QTL detected in identified plant.
In yet another aspect, feature of the present invention is the method for preparing F1 crossbred seed, the F1 crossbred plant that wherein derives from described F1 crossbred seed is anti-Sclerotinia, described method comprises plant or filial generation and the second plant cross-pollination that will identify according to method mentioned above, and wherein said the second plant lacks at least one allelotrope of at least one QTL detected in identified plant.
In yet another aspect, feature of the present invention is the method for the positional cloning nucleic acid that comprises the quantitative trait locus (QTL) be associated with the whole plant field resistance of Sclerotinia whole plant field resistance or raising, described method comprises: the nucleic acid that provides the plant of self-contained mark, described mark is associated with the whole plant field resistance of Sclerotinia whole plant field resistance or raising with the statistical significance of p≤0.01, wherein said QTL is positioned to and is selected from N1, N3, N4, N7, N8, N9, N10, N11, N12, N13, N15, the linkage group of N18 or N19, and wherein said linkage group comprises described mark, and clone comprise the quantitative trait locus (QTL) be associated with the whole plant field resistance of Sclerotinia whole plant field resistance or raising nucleic acid.(a) side joint comprise (i) mark CA0614 and PE0177 or (ii) interval of mark AG0093 and AG0482 on linkage group N1; (b) side joint the interval that comprises mark CA0410 and AG0023 on linkage group N3; (c) side joint the interval that comprises mark BG1442 and BG0106 on linkage group N4; (d) side joint the interval that comprises mark AG0510 and CA0105 on linkage group N7; (e) side joint the interval that comprises mark CA0837 and BG1286 on linkage group N8; (f) side joint comprise (i) mark CA1034 and AG0441 or (ii) interval of mark AG0378 and KK66 on linkage group N9; (g) side joint the interval that comprises mark BG0228 and PE0131 on linkage group N10; (h) side joint comprise (i) mark CA0120 and CA0163 or (ii) interval of mark CA0120 and CA1097 on linkage group N11; (i) side joint comprise (i) mark BG1321 and CA0991 or (ii) interval of mark CA0753 and PE0250 on linkage group N12; (j) side joint the interval that comprises mark CA0603 and CA0736 on linkage group N13; (k) side joint the interval that comprises mark PE0286 and AG0369 on linkage group N15; (l) side joint comprise (i) mark BG0278 and CA0636 or (ii) interval of mark UB0315 and CA0739 on linkage group N18; (m) side joint comprise (i) mark CA1107 and CA0221 or (ii) interval of mark UB0307 and KK98G on linkage group N19.
In another embodiment, described QTL is positioned to and is selected between following chromosomal region: (a) the one or more intervals on linkage group N1, and its side joint also comprises mark (i) AG0093 and PE0203 or (ii) BG0111 and BG1392 or (iii) BG1090 and AG0482 or (iv) BG1090 and PE0203 or (v) CA0614 and BG1392 or (vi) BG0988 and AG0482 or (vii) AG0243 and AG0482 or (viii) AG0243 and BG1453 or BG0988; (b) the one or more intervals on linkage group N3, its side joint also comprises mark (i) BG1197 and AG0023 or (ii) CA0410 and BG1368 or (iii) CA0410 and BG1197; (c) the one or more intervals on linkage group N4, its side joint also comprises mark (i) BG1442 and BG0106 or (ii) UB0181 and BG0106; (d) the one or more intervals on linkage group N8, its side joint also comprises mark (i) BG1449 and BG1062 or (ii) CA0837 and AG0328 or (iii) CA0837 and BG1062 or (iv) CA0837 and BG1101 or (v) CA0837 and BG1286 or (vi) CA0837 and BG1449 or (vii) PE0281 and BG0647; (e) the one or more intervals on linkage group N9, its side joint also comprises mark (i) AG0323 and BG0295 or (ii) CA1034 and AG0378 or (iii) BG1123 and AG0441; (f) the one or more intervals on linkage group N10, its side joint also comprises mark (i) BG0228 and AG0047 or BG0255 and PE0131; (g) the one or more intervals on linkage group N11, its side joint also comprises mark (i) BG0031 and BG1149 or (ii) BG0031 and BG1230 or (iii) BG0031 and BG1513 or (iv) CA0120 and CA0328 or (v) PE0283 and CA0163 or (vi) PE0324 and PE0283 or (vii) CA0328 and PE0324 or (viii) CA0226 and BG0713 or (ix) CA0233 and CA1080 or (x) CA0233 and AG0370; (h) the one or more intervals on linkage group N12, its side joint also comprises mark (i) BG1321 and CA0991 or (ii) BG1321 and CA1027 or (iii) BG1321 and PE0133 or (iv) PE0063 and CA0991 or (v) PE0133 and CA0991 or (vi) CA1027 and PE0063 or (vii) CA1027 and UB0331 or (viii) CA0423 and PE0250 or (ix) AG0359 and PE0250 or (x) AG0359 and CA0896; (i) the one or more intervals on linkage group N13, its side joint also comprises mark (i) BG0516 and AG0148 or (ii) CA0488 and AG0148 or (iii) CA0488 and CA0736 or (iv) CA0603 and AG0504 or (v) BG1288 and AG0504; (j) the one or more intervals on linkage group N15, its side joint also comprises mark (i) CA0719 and AG0369 or (ii) PE0091 and PE0187 or (iii) PE0286 and AG0369 or (iv) PE0286 and PE0187 or (v) PE0286 and CA0719; (k) the one or more intervals on linkage group N18, its side joint also comprises mark (i) AG0285 and CA0636 or (ii) BG0278 and CA07739 or (iii) CA0739 and CA0636 or (iv) UB0315 and CA0636 or (v) UB0315 and CA0739; (l) the one or more intervals on linkage group N19, its side joint also comprises mark (i) CA0552 and CA0221 or (ii) CA1107 and CA0552 or (iii) CA1107 and CA0221 or (iv) CA0221 and KK98G or (v) UB0307 and BG1241 or (vi) BG1241 and KK98G or (vii) CA0221 and BG1241.
In a specific embodiment, described QTL is positioned between the chromosomal region on linkage group N1, N9, N11, N12, N18 or N19.
In other embodiments, described mark Including Polymorphism, described polymorphism is by least one Identity of allele of described at least one quantitative trait locus (QTL) for to be associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising, and described detection comprises and identifies described polymorphism.Polymorphism can be, for example, and single nucleotide polymorphism (SNP) or simple repeated sequence (SSR).In another embodiment of method of the present invention, described detection comprises at least one mark of detection, and described mark is selected from: AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; CA1107; PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; And KK98G.
In another embodiment of method of the present invention, described detection comprises at least one mark of detection, and described mark is selected from: AG0093; AG0304; AG0378; AG0391; AG0482; BG1149; BG1230; BG1241; BG1453; BG1513; CA0120; CA0221; CA0546; CA0739; CA1027; PE0063; PE0203; UB0163; And UB0315.
In other embodiments, described plant is whole plant, plant organ, plant seed or vegetable cell.In other embodiments, described plant is that draw Kano.Described plant can be, for example, swede type rape (Brassica napus), leaf mustard (Brassica juncea), turnip (Brassica rapa), wild cabbage (Brassica oleracea) or brassicacarinata (Brassica carinata).Described plant can be that for example, draw the spring habit Kano, draw the winter habit Kano or draw the semi-winterness Kano.In another embodiment, described Sclerotinia whole plant field resistance plant is anti-sclerotinite.
In yet another aspect, feature of the present invention is the method for preparing the transgenosis dicotyledons that comprises the quantitative trait locus (QTL) be associated with the whole plant field resistance of Sclerotinia whole plant field resistance or raising, said method comprising the steps of: will import in the dicotyledons cell according to method clone's mentioned above nucleic acid; And described cell is grown under the Growth of Cells condition.In one embodiment, described QTL is positioned to and is selected between following chromosomal region: (a) side joint comprise (i) mark CA0614 and PE0177 or (ii) interval of mark AG0093 and AG0482 on linkage group N1; (b) side joint the interval that comprises mark CA0410 and AG0023 on linkage group N3; (c) side joint the interval that comprises mark BG1442 and BG0106 on linkage group N4; (d) side joint the interval that comprises mark AG0510 and CA0105 on linkage group N7; (e) side joint the interval that comprises mark CA0837 and BG1286 on linkage group N8; (f) side joint comprise (i) mark CA1034 and AG0441 or (ii) interval of mark AG0378 and KK66 on linkage group N9; (g) side joint the interval that comprises mark BG0228 and PE0131 on linkage group N10; (h) side joint comprise (i) mark CA0120 and CA0163 or (ii) interval of mark CA0120 and CA1097 on linkage group N11; (i) side joint comprise (i) mark BG1321 and CA0991 or (ii) interval of mark CA0753 and PE0250 on linkage group N12; (j) side joint the interval that comprises mark CA0603 and CA0736 on linkage group N13; (k) side joint the interval that comprises mark PE0286 and AG0369 on linkage group N15; (l) side joint comprise (i) mark BG0278 and CA0636 or (ii) interval of mark UB0315 and CA0739 on linkage group N18; (m) side joint comprise (i) mark CA1107 and CA0221 or (ii) interval of mark UB0307 and KK98G on linkage group N19.
In another embodiment, described QTL is positioned to and is selected between following chromosomal region: (a) the one or more intervals on linkage group N1, and its side joint also comprises mark (i) AG0093 and PE0203 or (ii) BG0111 and BG1392 or (iii) BG1090 and AG0482 or (iv) BG1090 and PE0203 or (v) CA0614 and BG1392 or (vi) BG0988 and AG0482 or (vii) AG0243 and AG0482 or (viii) AG0243 and BG1453 or BG0988; (b) the one or more intervals on linkage group N3, its side joint also comprises mark (i) BG1197 and AG0023 or (ii) CA0410 and BG1368 or (iii) CA0410 and BG1197; (c) the one or more intervals on linkage group N4, its side joint also comprises mark (i) BG1442 and BG0106 or (ii) UB0181 and BG0106; (d) the one or more intervals on linkage group N8, its side joint also comprises mark (i) BG1449 and BG1062 or (ii) CA0837 and AG0328 or (iii) CA0837 and BG1062 or (iv) CA0837 and BG1101 or (v) CA0837 and BG1286 or (vi) CA0837 and BG1449 or (vii) PE0281 and BG0647; (e) the one or more intervals on linkage group N9, its side joint also comprises mark (i) AG0323 and BG0295 or (ii) CA1034 and AG0378 or (iii) BG1123 and AG0441; (f) the one or more intervals on linkage group N10, its side joint also comprises mark (i) BG0228 and AG0047 or BG0255 and PE0131; (g) the one or more intervals on linkage group N11, its side joint also comprises mark (i) BG0031 and BG1149 or (ii) BG0031 and BG1230 or (iii) BG0031 and BG1513 or (iv) CA0120 and CA0328 or (v) PE0283 and CA0163 or (vi) PE0324 and PE0283 or (vii) CA0328 and PE0324 or (viii) CA0226 and BG0713 or (ix) CA0233 and CA1080 or (x) CA0233 and AG0370; (h) the one or more intervals on linkage group N12, its side joint also comprises mark (i) BG1321 and CA0991 or (ii) BG1321 and CA1027 or (iii) BG1321 and PE0133 or (iv) PE0063 and CA0991 or (v) PE0133 and CA0991 or (vi) CA1027 and PE0063 or (vii) CA1027 and UB0331 or (viii) CA0423 and PE0250 or (ix) AG0359 and PE0250 or (x) AG0359 and CA0896; (i) the one or more intervals on linkage group N13, its side joint also comprises mark (i) BG0516 and AG0148 or (ii) CA0488 and AG0148 or (iii) CA0488 and CA0736 or (iv) CA0603 and AG0504 or (v) BG1288 and AG0504; (j) the one or more intervals on linkage group N15, its side joint also comprises mark (i) CA0719 and AG0369 or (ii) PE0091 and PE0187 or (iii) PE0286 and AG0369 or (iv) PE0286 and PE0187 or (v) PE0286 and CA0719; (k) the one or more intervals on linkage group N18, its side joint also comprises mark (i) AG0285 and CA0636 or (ii) BG0278 and CA07739 or (iii) CA0739 and CA0636 or (iv) UB0315 and CA0636 or (v) UB0315 and CA0739; (l) the one or more intervals on linkage group N19, its side joint also comprises mark (i) CA0552 and CA0221 or (ii) CA1107 and CA0552 or (iii) CA1107 and CA0221 or (iv) CA0221 and KK98G or (v) UB0307 and BG1241 or (vi) BG1241 and KK98G or (vii) CA0221 and BG1241.
In a specific embodiment, described QTL is positioned between the chromosomal region on linkage group N1, N9, N11, N12, N18 or N19.
In other embodiments, described mark Including Polymorphism, described polymorphism is by least one Identity of allele of described at least one quantitative trait locus (QTL) for to be associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising, and described detection comprises and identifies described polymorphism.Polymorphism can be, for example, and single nucleotide polymorphism (SNP) or simple repeated sequence (SSR).In another embodiment of method of the present invention, described detection comprises at least one mark of detection, and described mark is selected from: AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; CA1107; PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; And KK98G.
In another embodiment, described detection comprises at least one mark of detection, and described mark is selected from: AG0093; AG0304; AG0378; AG0391; AG0482; BG1149; BG1230; BG1241; BG1453; BG1513; CA0120; CA0221; CA0546; CA0739; CA1027; PE0063; PE0203; UB0163; And UB0315.
In another embodiment, described dicotyledons cell is reproduced, to form the first plant.In another embodiment, the second plant hybridization of described the first plant and same species.In another embodiment, described dicotyledons is soybean, Sunflower Receptacle, draw Kano or clover.In another embodiment, described dicotyledons is that draw Kano, and for example, draw the spring habit Kano, draw the winter habit Kano or draw the semi-winterness Kano.In another embodiment, described dicotyledons is swede type rape (Brassica napus), leaf mustard (Brassica juncea), turnip (Brassica rapa) or wild cabbage (Brassica oleracea).In another embodiment, described Sclerotinia whole plant field resistance plant is anti-sclerotinite.In other embodiments, described whole plant field resistance is because the dicotyledons with lacking described QTL is compared the disease incidence of reduction or the disease severity of reduction.
The feature of another aspect of the present invention is to identify the method for the candidate nucleic acid from dicotyledons that comprises the QTL be associated with Sclerotinia whole plant field resistance, and described method comprises: the nucleic acid according to method clone mentioned above is provided; And the homologue of identifying nucleic acid described in dicotyledons.
The method of the feature of the another aspect of the present invention quantitative trait locus (QTL) that to be marker assisted selection (MAS) be associated with whole plant field resistance to Sclerotinia, said method comprising the steps of: obtain the first Brassica plants, described the first Brassica plants has at least one allelotrope of marker gene seat, and wherein said marker gene seat is associated with whole plant field resistance to Sclerotinia or the whole plant field resistance of raising with the statistical significance of p≤0.01; By described the first Brassica plants and the hybridization of the second Brassica plants; At least for described allelotrope assessment filial generation; And selection at least has described allelic progeny plant.In one embodiment, described plant is the member of segregating population.In another embodiment, described marker assisted selection is undertaken by high flux screening.
The feature of another aspect of the present invention is Brassica plants and its filial generation of identifying by method above, comprises F1, F2 and F3 filial generation.
The feature of another aspect of the present invention is nucleic acid that separate or restructuring, and it comprises and is selected from following polynucleotide: the sequence that is selected from any one following flag sequence: AG0023 (SEQ ID NO:1); AG0045 (SEQ ID NO:2); AG0047 (SEQ ID NO:3); AG0070 (SEQ ID NO:4); AG0086 (SEQ ID NO:5); AG0093 (SEQ ID NO:6); AG0125 (SEQ ID NO:7); AG0148 (SEQ ID NO:8); AG0171 (SEQ ID NO:9); AG0203 (SEQ ID NO:10); AG0239 (SEQ ID NO:11); AG0243 (SEQ ID NO:12); AG0272 (SEQ ID NO:13); AG0304 (SEQ ID NO:14); AG0323 (SEQ ID NO:15); AG0324 (SEQ ID NO:16); AG0328 (SEQ ID NO:17); AG0359 (SEQ ID NO:18); AG0369 (SEQ ID NO:19); AG0370 (SEQ ID NO:20); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0410 (SEQ ID NO:23); AG0441 (SEQ ID NO:24); AG0477 (SEQ ID NO:25); AG0482 (SEQ ID NO:26); AG0504 (SEQ ID NO:27); AG0510 (SEQ ID NO:28); BG0031 (SEQ ID NO:29); BG0106 (SEQ ID NO:30); BG0111 (SEQ ID NO:31); BG0119 (SEQ ID NO:32); BG0181 (SEQ ID NO:33); BG0228 (SEQ ID NO:34); BG0255 (SEQ ID NO:35); BG0278 (SEQ ID NO:36); BG0295 (SEQ ID NO:37); BG0452 (SEQ ID NO:38); BG0516 (SEQ ID NO:39); BG0647 (SEQ ID NO:40); BG0651 (SEQ ID NO:41); BG0713 (SEQ ID NO:42); BG0864 (SEQ ID NO:43); BG0869 (SEQ ID NO:44); BG0988 (SEQ ID NO:45); BG1062 (SEQ ID NO:46); BG1090 (SEQ ID NO:47); BG1101 (SEQ ID NO:48); BG1123 (SEQ ID NO:49); BG1127 (SEQ ID NO:50); BG1149 (SEQ ID NO:51); BG1182 (SEQ ID NO:52); BG1197 (SEQ ID NO:53); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1244 (SEQ ID NO:56); BG1286 (SEQ ID NO:57); BG1288 (SEQ ID NO:58); BG1321 (SEQ ID NO:59); BG1368 (SEQ ID NO:60); BG1392 (SEQ ID NO:61); BG1442 (SEQ ID NO:62); BG1449 (SEQ ID NO:63); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); CA0105 (SEQ ID NO:66); CA0120 (SEQ ID NO:67); CA0163 (SEQ ID NO:68); CA0221 (SEQ ID NO:69); CA0226 (SEQ ID NO:70); CA0233 (SEQ ID NO:71); CA0328 (SEQ ID NO:72); CA0410 (SEQ ID NO:73); CA0423 (SEQ ID NO:74); CA0456 (SEQ ID NO:75); CA0488 (SEQ ID NO:76); CA0546 (SEQ ID NO:77); CA0552 (SEQ ID NO:78); CA0603 (SEQ ID NO:79); CA0614 (SEQ ID NO:80); CA0636 (SEQ ID NO:81); CA0681 (SEQ ID NO:82); CA0719 (SEQ ID NO:83); CA0736 (SEQ ID NO:84); CA0739 (SEQ ID NO:85); CA0753 (SEQ ID NO:86); CA0834 (SEQ ID NO:87); CA0837 (SEQ ID NO:88); CA0896 (SEQ ID NO:89); CA0991 (SEQ ID NO:90); CA1027 (SEQ ID NO:91); CA1032 (SEQ ID NO:92); CA1034 (SEQ ID NO:93); CA1035 (SEQ ID NO:94); CA1066 (SEQ ID NO:95); CA1080 (SEQ ID NO:96); CA1090 (SEQ ID NO:97); CA1097 (SEQ ID NO:98); Or CA1107 (SEQ ID NO:99); (b) there is the polynucleotide sequence of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with polynucleotide (a); (c) with (a) or the polynucleotide sequence of polynucleotide sequence complementation (b).In one embodiment, described separation or nucleic acid restructuring is associated with the whole plant field resistance to Sclerotinia.
In another embodiment, described separation or nucleic acid restructuring comprises and is selected from following polynucleotide: the sequence that (a) is selected from any one following flag sequence: AG0093 (SEQ ID NO:6); AG0304 (SEQ ID NO:14); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0482 (SEQ ID NO:26); BG1149 (SEQ ID NO:51); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); (SEQ ID NO:67); CA0221 (SEQ ID NO:69); CA0546 (SEQ ID NO:77); CA0739 (SEQ ID NO:85); Or CA1027 (SEQ ID NO:91); (b) there is the polynucleotide sequence of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with polynucleotide (a); (c) with (a) or the polynucleotide sequence of polynucleotide sequence complementation (b).
The feature of another aspect of the present invention is the nucleic acid molecule separated for detection of the polymorphism be associated with the whole plant field resistance of whole plant field resistance to Sclerotinia or raising in DNA of plants, wherein said nucleic acid molecule comprises at least 15 Nucleotide, and be identical with the sequence of the continuous nucleotide of arbitrary chain equal amts of the DNA of plants in zone that described polymorphism is arranged in, wherein said nucleic acid molecule comprises the fragment at least 70% with the flag sequence that is selected from following mark or flag sequence, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequence: AG0023 (SEQ ID NO:1), AG0045 (SEQ ID NO:2), AG0047 (SEQ ID NO:3), AG0070 (SEQ ID NO:4), AG0086 (SEQ ID NO:5), AG0093 (SEQ ID NO:6), AG0125 (SEQ ID NO:7), AG0148 (SEQ ID NO:8), AG0171 (SEQ ID NO:9), AG0203 (SEQ ID NO:10), AG0239 (SEQ ID NO:11), AG0243 (SEQ ID NO:12), AG0272 (SEQ ID NO:13), AG0304 (SEQ ID NO:14), AG0323 (SEQ ID NO:15), AG0324 (SEQ ID NO:16), AG0328 (SEQ ID NO:17), AG0359 (SEQ ID NO:18), AG0369 (SEQ ID NO:19), AG0370 (SEQ ID NO:20), AG0378 (SEQ ID NO:21), AG0391 (SEQ ID NO:22), AG0410 (SEQ ID NO:23), AG0441 (SEQ ID NO:24), AG0477 (SEQ ID NO:25), AG0482 (SEQ ID NO:26), AG0504 (SEQ ID NO:27), AG0510 (SEQ ID NO:28), BG0031 (SEQ ID NO:29), BG0106 (SEQ ID NO:30), BG0111 (SEQ ID NO:31), BG0119 (SEQ ID NO:32), BG0181 (SEQ ID NO:33), BG0228 (SEQ ID NO:34), BG0255 (SEQ ID NO:35), BG0278 (SEQ ID NO:36), BG0295 (SEQ ID NO:37), BG0452 (SEQ ID NO:38), BG0516 (SEQ ID NO:39), BG0647 (SEQ ID NO:40), BG0651 (SEQ ID NO:41), BG0713 (SEQ ID NO:42), BG0864 (SEQ ID NO:43), BG0869 (SEQ ID NO:44), BG0988 (SEQ ID NO:45), BG1062 (SEQ ID NO:46), BG1090 (SEQ ID NO:47), BG1101 (SEQ ID NO:48), BG1123 (SEQ ID NO:49), BG1127 (SEQ ID NO:50), BG1149 (SEQ ID NO:51), BG1182 (SEQ ID NO:52), BG1197 (SEQ ID NO:53), BG1230 (SEQ ID NO:54), BG1241 (SEQ ID NO:55), BG1244 (SEQ ID NO:56), BG1286 (SEQ ID NO:57), BG1288 (SEQ ID NO:58), BG1321 (SEQ ID NO:59), BG1368 (SEQ ID NO:60), BG1392 (SEQ ID NO:61), BG1442 (SEQ ID NO:62), BG1449 (SEQ ID NO:63), BG1453 (SEQ ID NO:64), BG1513 (SEQ ID NO:65), CA0105 (SEQ ID NO:66), CA0120 (SEQ ID NO:67), CA0163 (SEQ ID NO:68), CA0221 (SEQ ID NO:69), CA0226 (SEQ ID NO:70), CA0233 (SEQ ID NO:71), CA0328 (SEQ ID NO:72), CA0410 (SEQ ID NO:73), CA0423 (SEQ ID NO:74), CA0456 (SEQ ID NO:75), CA0488 (SEQ ID NO:76), CA0546 (SEQ ID NO:77), CA0552 (SEQ ID NO:78), CA0603 (SEQ ID NO:79), CA0614 (SEQ ID NO:80), CA0636 (SEQ ID NO:81), CA0681 (SEQ ID NO:82), CA0719 (SEQ ID NO:83), CA0736 (SEQ ID NO:84), CA0739 (SEQ ID NO:85), CA0753 (SEQ ID NO:86), CA0834 (SEQ ID NO:87), CA0837 (SEQ ID NO:88), CA0896 (SEQ ID NO:89), CA0991 (SEQ ID NO:90), CA1027 (SEQ ID NO:91), CA1032 (SEQ ID NO:92), CA1034 (SEQ ID NO:93), CA1035 (SEQ ID NO:94), CA1066 (SEQ ID NO:95), CA1080 (SEQ ID NO:96), CA1090 (SEQ ID NO:97), CA1097 (SEQ ID NO:98), or CA1107 (SEQ ID NO:99).In one embodiment, described nucleic acid is selected from any SEQ ID NO:126-323.
In another embodiment, described flag sequence is: AG0093 (SEQ ID NO:6); AG0304 (SEQ ID NO:14); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0482 (SEQ ID NO:26); BG1149 (SEQ ID NO:51); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); (SEQ ID NO:67); CA0221 (SEQ ID NO:69); CA0546 (SEQ ID NO:77); CA0739 (SEQ ID NO:85); Or CA1027 (SEQ ID NO:91).In one embodiment, described nucleic acid is selected from any SEQ ID NO:136,137,152,153,166,167,168,169,176,177,226,227,232,233,234,235,252,253,254,255,258,259,262,263,278,279,294,295,306 or 307.
The feature of another aspect of the present invention is the QTL screening plant that is associated for the whole plant field resistance for to Sclerotinia or the test kit of germplasm, described test kit comprises container, comprises the nucleic acid molecule of the separation of one or more claims as described above in described container; With the be associated specification sheets of QTL screening plant of the whole plant field resistance of whole plant field resistance for for to Sclerotinia or raising.
In one embodiment, described test kit also comprises damping fluid.In another embodiment, described test kit is for high flux screening, and comprises at least one component for this type of purposes.In another embodiment, described test kit is in the high flux screening of integration system.
In yet another aspect, feature of the present invention is the Brassica plants shown the whole plant field resistance of the whole plant field resistance of Sclerotinia or raising, described plant includes the allelotrope that is beneficial to Sclerotinia whole plant field resistance at least 1 QTL, described QTL is positioned to and is selected between following chromosomal region: (a) side joint comprise (i) mark CA0614 and PE0177 or (ii) interval of mark AG0093 and AG0482 on linkage group N1; (b) side joint the interval that comprises mark CA0410 and AG0023 on linkage group N3; (c) side joint the interval that comprises mark BG1442 and BG0106 on linkage group N4; (d) side joint the interval that comprises mark AG0510 and CA0105 on linkage group N7; (e) side joint the interval that comprises mark CA0837 and BG1286 on linkage group N8; (f) side joint comprise (i) mark CA1034 and AG0441 or (ii) interval of mark AG0378 and KK66 on linkage group N9; (g) side joint the interval that comprises mark BG0228 and PE0131 on linkage group N10; (h) side joint comprise (i) mark CA0120 and CA0163 or (ii) interval of mark CA0120 and CA1097 on linkage group N11; (i) side joint comprise (i) mark BG1321 and CA0991 or (ii) interval of mark CA0753 and PE0250 on linkage group N12; (j) side joint the interval that comprises mark CA0603 and CA0736 on linkage group N13; (k) side joint the interval that comprises mark PE0286 and AG0369 on linkage group N15; (l) side joint comprise (i) mark BG0278 and CA0636 or (ii) interval of mark UB0315 and CA0739 on linkage group N18; (m) side joint comprise (i) mark CA1107 and CA0221 or (ii) interval of mark UB0307 and KK98G on linkage group N19.
In another embodiment, described QTL is positioned to and is selected between following chromosomal region: (a) the one or more intervals on linkage group N1, and its side joint also comprises mark (i) AG0093 and PE0203 or (ii) BG0111 and BG1392 or (iii) BG1090 and AG0482 or (iv) BG1090 and PE0203 or (v) CA0614 and BG1392 or (vi) BG0988 and AG0482 or (vii) AG0243 and AG0482 or (viii) AG0243 and BG1453 or BG0988; (b) the one or more intervals on linkage group N3, its side joint also comprises mark (i) BG1197 and AG0023 or (ii) CA0410 and BG1368 or (iii) CA0410 and BG1197; (c) the one or more intervals on linkage group N4, its side joint also comprises mark (i) BG1442 and BG0106 or (ii) UB0181 and BG0106; (d) the one or more intervals on linkage group N8, its side joint also comprises mark (i) BG1449 and BG1062 or (ii) CA0837 and AG0328 or (iii) CA0837 and BG1062 or (iv) CA0837 and BG1101 or (v) CA0837 and BG1286 or (vi) CA0837 and BG1449 or (vii) PE0281 and BG0647; (e) the one or more intervals on linkage group N9, its side joint also comprises mark (i) AG0323 and BG0295 or (ii) CA1034 and AG0378 or (iii) BG1123 and AG0441; (f) the one or more intervals on linkage group N10, its side joint also comprises mark (i) BG0228 and AG0047 or BG0255 and PE0131; (g) the one or more intervals on linkage group N11, its side joint also comprises mark (i) BG0031 and BG1149 or (ii) BG0031 and BG1230 or (iii) BG0031 and BG1513 or (iv) CA0120 and CA0328 or (v) PE0283 and CA0163 or (vi) PE0324 and PE0283 or (vii) CA0328 and PE0324 or (viii) CA0226 and BG0713 or (ix) CA0233 and CA1080 or (x) CA0233 and AG0370; (h) the one or more intervals on linkage group N12, its side joint also comprises mark (i) BG1321 and CA0991 or (ii) BG1321 and CA1027 or (iii) BG1321 and PE0133 or (iv) PE0063 and CA0991 or (v) PE0133 and CA0991 or (vi) CA1027 and PE0063 or (vii) CA1027 and UB0331 or (viii) CA0423 and PE0250 or (ix) AG0359 and PE0250 or (x) AG0359 and CA0896; (i) the one or more intervals on linkage group N13, its side joint also comprises mark (i) BG0516 and AG0148 or (ii) CA0488 and AG0148 or (iii) CA0488 and CA0736 or (iv) CA0603 and AG0504 or (v) BG1288 and AG0504; (j) the one or more intervals on linkage group N15, its side joint also comprises mark (i) CA0719 and AG0369 or (ii) PE0091 and PE0187 or (iii) PE0286 and AG0369 or (iv) PE0286 and PE0187 or (v) PE0286 and CA0719; (k) the one or more intervals on linkage group N18, its side joint also comprises mark (i) AG0285 and CA0636 or (ii) BG0278 and CA07739 or (iii) CA0739 and CA0636 or (iv) UB0315 and CA0636 or (v) UB0315 and CA0739; (l) the one or more intervals on linkage group N19, its side joint also comprises mark (i) CA0552 and CA0221 or (ii) CA1107 and CA0552 or (iii) CA1107 and CA0221 or (iv) CA0221 and KK98G or (v) UB0307 and BG1241 or (vi) BG1241 and KK98G or (vii) CA0221 and BG1241.
In a specific embodiment, described QTL is positioned between the chromosomal region on linkage group N1, N9, N11, N12, N18 or N19.
Other features and advantages of the present invention will be understood by summary of the invention and example hereinafter.
Embodiment
summary
(for example the present invention relates to the genetic marker relevant to Sclerotinia whole plant field resistance, the marker gene seat and corresponding to (or deriving from) these the nucleic acid of marker gene seat, probe and amplified production as useful as the gene type for to plant) evaluation.Mark of the present invention is used to identify the plant to plant, especially swede type rape (Brassica napus (B.napus)) (draw Kano) species of the Sclerotinia resistance or resistance that show raising.Therefore, these marks are for marker assisted selection (MAS) and the breeding of Sclerotinia resistance plant, and are useful for the evaluation of susceptible plants.Mark of the present invention also be used to identify and limit the contiguous quantitative trait locus be associated with Sclerotinia whole plant field resistance nucleic acid and/or corresponding to or comprise between the chromosomal region of described quantitative trait locus.The quantitative trait locus (QTL) be associated with Sclerotinia whole plant field resistance is interval by the heredity to being limited by a pair of mark as herein described or is limited and comprised the positional cloning separation of the nucleic acid of the nucleic acid of subsequence in interval of this type of mark or contiguous described hereditary interval or subsequence by this class mark.The preparation table that can be used to the QTL nucleic acid of this type of separation reveals transgenic cell and the plant to the resistance of the raising of Sclerotinia.In addition, from a kind of biology (for example, draw Kano) the QTL nucleic acid that separates, can then be used for (comprising multiple commercially important dicotyledons from other susceptible is biological, for example soybean, clover, Sunflower Receptacle, flax, beans (for example, white peas or beans), potato, pea and peanut) Separated pin is to the homologue of the QTL of Sclerotinia whole plant field resistance.
definition
The form that unit, prefix and symbol are accepted with their International System of Units (SI) means.Unless otherwise indicated, nucleic acid be with 5 ' to 3 ' direction, write from left to right; Aminoacid sequence is respectively to write from left to right to the carboxyl direction with amino.The numerical range of mentioning in this specification sheets comprises the numeral that limits this scope, and is included in each integer in this limited range.Nucleotide can be recommended with IUPAC-IUBMB NK in this article, and their one-letter symbol means.Specification sheets is done when as a whole, and the term below defined just defines more comprehensively.The chapter title that this specification sheets provides in the whole text is to provide for convenience's sake, is not the restriction to various objects of the present invention and embodiment.
Term " Sclerotinia whole plant field resistance " or " to the whole plant field resistance of Sclerotinia ", refer under field condition or under extreme disease pressure field research condition (as, for example, described in this paper and WO 2006/135717), the resistance of plant to the phytopathogen Sclerotinia.It has reflected the resistance of whole plant when being exposed to Sclerotinia under these conditions.In one embodiment, based on sclerotinite morbidity severity (SSDIS) scale, the plant with Sclerotinia whole plant field resistance has 5.0 or higher disease development scoring.In other embodiments, based on sclerotinite morbidity severity (SSDIS) scale, the plant with Sclerotinia whole plant field resistance has 5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0 or higher disease development scoring.The scoring of disease development is sometimes with Range Representation; For example, with the scope of 5-6,6-7,7-8,8-9 or with the scope of 5-7,7-9 etc., or with the numerical range within integer, for example, 5.5-6.5,5.5-7.5,6-7.5,7-8.5.In these examples, based on sclerotinite morbidity severity (SSDIS) scale, the plant with Sclerotinia whole plant field resistance has the disease development scoring of 5-6 at least or 6-7 or 7-8 or 8-9 scope.
The technician will know, the favourable allelic quantity (or per-cent) for Sclerotinia whole plant field resistance that plants has is higher, and the resistance level shown will be higher.Table 8 with reference to this paper can be understood this concept.In certain embodiments, the plant that has a Sclerotinia whole plant field resistance has the genome comprised at least about 50% favorable allels.In embodiment more specifically, plant with Sclerotinia whole plant field resistance has and comprises at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or the genome of more favorable allels.The per-cent of favorable allels can also be expressed as numerical value.For example, as shown in table 8, if add up to the favorable allels of 15, in certain mapping population, be possible, there is the plant of 12 in these allelotrope and will there is 80% favorable allels.In certain embodiments, in plant the quantity of favorable allels or percentage ratio can be as plant the rough prediction index by the Sclerotinia whole plant field resistance level of the expection that shows.
The technician also will know, QTL described herein has represented the genome area of the gene that includes the Sclerotinia whole plant field resistance that helps plant.In addition, each QTL can differently contribute to this resistance level.Therefore, breeding work has pointed to the quantity that improves these QTL that exist in germplasm, especially quantitatively important QTL.The procedure of breeding early stage, may have less QTL in specific germplasm, but this quantity will improve along with the carrying out of the procedure of breeding.Therefore, in certain embodiments, the plant that shows Sclerotinia whole plant field resistance can comprise at least 6 QTL as herein described.More specifically, described plant can comprise at least 7,8,9 or 10 QTL as herein described.More specifically, described plant can comprise 11,12 or whole QTL as herein described.
In the present invention; assessment to the whole plant field resistance is used; for example, extreme disease pressure field research condition, simulated the grower's who draws Kano field condition and the land for growing field crops situation of worst case (its need to use two kinds of mycocides come cover crop to avoid Sclerotinia infect).
Term " Sclerotinia improve whole plant field resistance " or " to the whole plant field resistance of the raising of Sclerotinia ", refer under field condition or under extreme disease pressure field research condition (as, for example, described in this paper and WO 2006/135717), the raising of plant to the resistance of phytopathogen Sclerotinia.In one embodiment, there is the plant to the whole plant field resistance of the raising of Sclerotinia, be and do not there is described at least one allelic plant and compare, there is at least one allelotrope of the QTL be associated with whole plant field resistance to Sclerotinia, and have 5.0 or the plant of the higher disease development scoring based on the SSDIS scale.Other embodiment of the whole plant field resistance that Sclerotinia improves is similar to those that summarize in the above listed description to Sclerotinia whole plant field resistance.
Sclerotinia affects the many different tissue of plant.The natural infection of Sclerotinia starts from the infection of petal.When infected petal drops on leaf, disease is transmitted to leaf.Then, pathology development simultaneously on many blades on each plant.These pathologies are expanded and make blade withered to blade, infect by petiole and further develop towards stem simultaneously.Then, infect and arrive stem, and further develop in stem, cause too early maturation.
Having the plant to the whole plant field resistance of Sclerotinia, is the plant of the approach of anti-Sclerotinia disease development in whole tissues of this plant.Therefore, the plant had the whole plant field resistance of Sclerotinia also can be called as the plant had " approach resistance " or " the land for growing field crops approach resistance " of Sclerotinia.Screening method described herein is used to identify to have the plant to the whole plant field resistance of Sclerotinia.With other screening method as known in the art of resistance for assessment of to Sclerotinia, compare, these methods are special.Only detect the part of plant for assessment of other screening method as known in the art of the resistance to Sclerotinia, and these methods are to carry out with the unconnected growth phase of the natural development of disease.For example, the people such as Zhou (2003, TAG 106:759-764) are by having carried out phenotype analytical at seedling stage inoculation leaf with by the ripe plant stage, inoculating stem.The people such as Zhou (2006, TAG 112:509-5160) based on petiole, inoculation has been carried out phenotype analytical to the single plant of each strain, and the people such as Bela (17th Crucifer Genetics Workshop (Brassica 2010), in September, 2010, Saskatoon, Canada) inoculate 12 plant of each strain have been carried out to phenotype analytical based on petiole.The people such as Yin (2010, Euphytica, online version: DOI 10.1007/s10681-009-0095-1) adopted three kinds of inoculation methods: the petal inoculation that the inoculation of mycelium toothpick, the mycelium tamper on stem leaf inoculated and infected.Although use this type of screening method likely to identify one or more QTL that are associated with the Sclerotinia resistance, these screening methods are unlike identifying as described herein comprehensive the screening method of the whole plant field resistance of Sclerotinia.Although this means that these other screening methods can be used to be disclosed in the particular organization of plant one or a few QTL be associated with resistance to Sclerotinia, they can not be used to identify the whole QTL that are associated with the Sclerotinia resistance that spread all over whole plant.In addition, relate to single screening method of planting tissue rather than whole plant, the gene that can not detect in different tissues concurs to affect the epistatic effect that the Sclerotinia resistance causes.
The resistance detected Sclerotinia by the whole plant method has multiple advantage.At first, the method the most closely is similar to the natural interaction large Tanaka of Sclerotinia and plant, and should be therefore the system of identifying therein the excellence of the QTL be associated with resistance to Sclerotinia.The second, with respect to other screening method that only detects a kind of plant tissue, the whole plant method allows the evaluation to the QTL of greater amt.The 3rd, be different from other screening method, the method allows the analysis to epistatic effect.The 4th, the method allows the land for growing field crops performance from its data prediction reality.
Term " quantitative trait locus " or " QTL " refer to have the allelic polymorphism genetic loci that at least two differences ground affects the phenotypic character (for example,, to the whole plant field resistance of Sclerotinia or to the whole plant field resistance of the raising of Sclerotinia) of continuous distribution.For example, QTL can have favourable allelotrope, and it is given or contributes to the whole plant field resistance of Sclerotinia or to the whole plant field resistance of the raising of Sclerotinia.
Term " favourable allelotrope " is the allelotrope at the specific gene seat, it is given or contributes to desired phenotype, for example, to the whole plant field resistance of Sclerotinia or to the whole plant field resistance of the raising of Sclerotinia, perhaps alternatively, be the allelotrope of evaluation that allows the plant of the whole plant field resistance to having reduction, described plant can be removed (" the anti-selection ") from the procedure of breeding or plantation.The favorable allels of mark be with favourable phenotype be divided into from marker allele, or alternatively, with unfavorable plant phenotype be divided into from, thereby the beneficial effect of plant identification is provided.For example, in table 7 and table 13, provide for the whole plant field resistance to Sclerotinia or to the favourable allelotrope of whole plant field resistance of the raising of Sclerotinia.
Term " with being associated " or " being associated " refer in the context of the present invention, for example, nucleic acid in linkage disequilibrium and phenotypic character or the second nucleic acid,, with described nucleic acid, with the situation that phenotype/the second nucleic acid separates respectively, compare, described nucleic acid be present in progeny plant together with described proterties/second nucleic acid is found more frequently.
Term " chain " for example, for describing a marker gene seat and another marker gene seat or some other locus (degree, QTL) " be associated ".Linkage relationship between molecule marker and phenotype means with " probability " or " adjustment probability ".Chain can the expression by limit or the scope of expectation.For example, in certain embodiments, when mark, to be less than 50,40,30,25,20 or 15 collection of illustrative plates units (or cM) when separated, any mark (in heredity and physically) is chain with any other mark.In some respects, it is favourable limiting bracketed chain scope, for example between 10 and 20cM between, between 10 and 30cM between, or between 10 and 40cM between.Mark and the second locus chain tightr, mark is better to the indicating effect of the second locus.Therefore, " closely linked locus " is as marker gene seat and the second locus demonstration 10% or lower, preferably approximately 9% or lower, more preferably from about 8% or lower, more preferably from about 7% or lower, more preferably from about 6% or lower, more preferably from about 5% or lower, more preferably from about 4% or lower, more preferably from about 3% or lower, more preferably from about 2% or lower locus in recombination frequency.In highly preferred embodiment, the associated gene seat shows approximately 1% or lower recombination frequency, for example approximately 0.75% or lower, more preferably from about 0.5% or lower, more preferably from about 0.25% or lower recombination frequency.Be positioned on the phase homologous chromosomes, and the distance between them makes two restructuring occurrence frequencies between locus be less than 10% two locus of (for example approximately 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or lower) be also referred to as to be each other " vicinity " or " being adjacent to " each other.Because 1cM is the distance demonstrated between two marks of 1% recombination frequency, any other mark close linkage of any mark and next-door neighbour (on gene and physically), for example be equal to or less than the distance of 10cM.Two closely linked marks on the phase homologous chromosomes can be by mutually positioning in 9,8,7,6,5,4,3,2,1,0.75,0.5 or 0.25cM or less.
Term " linkage disequilibrium " refers to the nonrandom separation of locus.This means that this type of locus is physically contiguous fully along chromosomal length, make them tend to be greater than random frequency and jointly separate.
Term " chain in heredity " refers in linkage disequilibrium and on statistics and is determined the locus that can not combine independently.In the time of from 51% to 99% or between any round values (preferably at least 60%, 70%, 80%, 90%, 95% or 99%), locus chain in heredity combines relatively.The locus of heredity or allelotrope are known as chainly by this way, and are known as " linkage group ".
" probable value " or " P value " is the existence of the particular combination of phenotype and specific markers or not exist be random statistics probability.Probable value is lower, phenotype and attribute tags will be divided into from possibility larger.In some aspects, probable value is considered to " significantly " or " inapparent ".In certain embodiments, think the probable value of random separation be 0.05 (p=0.05, or 5% probability) be divided into from the significance indication.Yet acceptable probability can be any probability that is less than 50% (p=0.5).For example, significantly probability can be less than 0.25, is less than 0.2, is less than 0.15, is less than 0.1, is less than 0.05, is less than 0.01 or be less than 0.001.
Term " marker gene seat " is can find therein the specific chromosome position of specific markers in the species gene group.What " marker gene seat " can be used for follow the trail of existing second connects locus, for example encodes or contributes to express the connection locus of phenotypic character.For example the marker gene seat can be used in monitoring allelotrope in the separation of a certain locus, for example QTL or single-gene, and they and marker gene seat are in heredity or chain physically.
Term " mark " is nucleotide sequence or its coded product (for example protein) as reference point.For the mark for detection of restructuring, they need to be in monitored population checkout discrepancy or polymorphism.For molecule marker, this for example refers to, owing to the difference of the DNA level of polynucleotide sequence difference (, SSR, RFLP, FLP, SNP).The genome mutability can be any origin, for example inserts, lacks, copies, existence and the sequence of repeat element, point mutation, recombination event or transposable element.Molecule marker can derive from the nucleic acid (for example EST) of genome or expression, and also can refer to the nucleic acid as probe or primer pair, and described primer pair can be by being used the method extension increasing sequence fragment of PCR-based.A large amount of Btassica molecule markers are known in the art, and be announced or can obtain from multiple source.
The example of mark is provided in SEQ ID NO:1-125.It should be appreciated by those skilled in the art, mark of the present invention can comprise the complete sequence of any one sequence listed in SEQ ID NO:1-125 or the fragment of such sequence.Described fragment can be, for example, SSR (as listed at table 14 for example, or the sequence of flank (for example, as listed as SEQ ID NO:126-325 those) and comprise as described in SSR.Those skilled in the art be also to be understood that mark as the sequence of listed those in any SEQ ID NO:1-125 or the fragment of sequence will there are some variations between strain and strain.Therefore, mark of the present invention comprises the sequence that 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity is provided with sequence as provided in any SEQ ID NO:1-125 or its fragment.
The method that can set up by this area corresponding to the mark of the genetic polymorphism between kind of group members is detected.These methods comprise that sequence-specific amplification method, the restriction fragment length polymorphism of for example DNA sequencing, PCR-based detect that (RFLP), isozyme mark detect, the amplification variable sequence of by allele-specific, hybridize polynucleotide polymorphic detection that (ASH) carry out, Plant Genome detects, self-sustained sequence replication detects, simple repeated sequence detects (SSR), single nucleotide polymorphism detects (SNP) or amplified fragment length polymorphism detection (AFLP).The method of having set up also becomes known for the SSR mark that detects expressed sequence tag (EST) and derive from est sequence and the polymorphic dna (RAPD) of random amplification.
When identifying the linked gene seat, term " molecule marker " can be used for referring to the mark based on nucleic acid of any type, or it for example, as the coded product (, protein) of reference point.Mark can derive from genome nucleotide sequence or derive from the nucleotide sequence of expression (such as the RNA that derives from montage, cDNA etc.), or derives from the polypeptide of coding.This term also refers to the nucleotide sequence with flag sequence complementation or side joint flag sequence, as the nucleic acid as probe or primer pair that can the amplification label sequence." molecular mark probe " is nucleotide sequence or the molecule that can be used to the existence of identifying mark locus, for example, with the nucleic acid probe of marker gene seat sequence complementation.Alternatively, molecular mark probe refers to distinguish the probe that (being gene type) is present in the specific allelic any type on the marker gene seat in some aspects.When nucleic acid, in solution during specific hybrid, for example, according to the hybridization of Watson-Crick basepairing rule, nucleic acid is " complementation ".Any suitable mark detection technique all can be used for identifying this type of hybridization mark, and for example, the SSR technology is used to example provided herein.
" marker allele " or " allelotrope of marker gene seat " can refer in population to be positioned at one of them of a plurality of polymorphic nucleotide sequences of marker gene seat, and it is polymorphic with regard to the marker gene seat.
Term " interval " refers to the continuous linearity range of the chromosomal DNA with terminal, and it is usually limited by molecule marker and comprises molecule marker.
Term " nucleic acid ", " Nucleotide ", " polynucleotide ", " polynucleotide sequence " and " nucleotide sequence " refer to strand or double-stranded thymus nucleic acid or Yeast Nucleic Acid polymkeric substance or their block polymer.As used herein, this term can be additionally or is comprised alternatively the analogue of naturally occurring Nucleotide, described analogue has the essential property (for example, peptide nucleic acid(PNA)) of natural nucleotide aspect the mode with similar to naturally occurring Nucleotide and single-chain nucleic acid hybridization.Except as otherwise noted, except the sequence explicitly pointed out, specific nucleic acid sequence of the present invention is also optionally contained complementary sequence.Term " gene " is used to finger, for example, and cDNA and the mRNA encoded by genome sequence, and this genome sequence.
Term " homology " refers to that nucleotide sequence derives from common ancestral gene (for example, being the member of identical gene family) by natural or artificial process, and therefore usually has sequence similarity.Usually, homologous nucleic acid has sufficient sequence identity, and one of calling sequence or its complementary sequence can be hybridized with another sequence selective ground under the selective cross condition.Term " selective cross " comprises and referring under stringent hybridization condition, but the detection level that nucleotide sequence and specific nucleic acid target sequence are higher with the hybridization than itself and non-target nucleic acid sequence (for example at least 2 times to background) hybridization, and refer to substantially get rid of non-target nucleic acid.The sequence of selective cross has approximately at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity each other.Show with the nucleic acid of the homology of at least some degree of reference nucleic acid can be with this reference nucleic acid or its complementary sequence identical or it is distinctive.
Term " separation " refers to material, and for example nucleic acid or protein are free in its naturally occurring environment usually basically with together with it or component interactional with it.The material separated optionally comprises not for example, in the natural surroundings (, cell) of this material found material.In addition, for example, if described material is in its natural surroundings (cell), described material has been placed in the position (for example, genome or subcellular organelle) in the non-natural cell of material for existing in this environment.For example, naturally occurring nucleic acid (for example, promotor), if its mode existed by non-natural is imported into the locus in the non-natural genome of this nucleic acid, be considered to separate." separation " nucleic acid, also be known as " allos " nucleic acid as herein defined.
Term " restructuring " means that this material (for example, nucleic acid or protein) is synthesized ground (non-natural ground) by manual intervention and changes.Can or make it break away from natural surroundings or state under the natural surroundings of material or state, described material is changed to produce synthetic material.For example, if make naturally occurring nucleic acid be changed by manual intervention in the natural cell that exists nucleic acid to be derived from, or change occurred in the DNA that transcribes out this nucleic acid, and this naturally occurring nucleic acid is considered to recombinant nucleic acid.Referring to, for example, Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, United States Patent (USP) 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; The people such as Zarling, PCT/US93/03868.
Term " importing " is when referring to nucleic acid allos or that separate, refer to that nucleic acid is to mixing in eucaryon or prokaryotic cell prokaryocyte, wherein said nucleic acid (for example can be integrated in the genome of this cell in described cell, karyomit(e), plasmid, plastid or Mitochondrial DNA), be transformed in self-replicating or express instantaneously (for example, the mRNA of transfection).This term comprises that the method that imports nucleic acid is as " transfection ", " conversion " and " transduction ".
Term " SSR " or " simple repeated sequence " refer to and are present in the pleomorphism site that the repeating unit by 1-6 base pair length in core or organelle DNA forms.Different allelotrope can have the repetition SSR of different quantities, causes allelic different lengths, as passed through, for example, the detected through gel electrophoresis after the described allelotrope of amplification.For example, it can be GAGAGAGA that dinucleotides repeats, and the trinucleotide repetition can be ATGATGATGATG.It is believed that when DNA is replicated, can make a mistake in this process, and the extra group of these tumor-necrosis factor glycoproteinss is added in chain.Along with time lapse, difference occurs in these tumor-necrosis factor glycoproteinss between a Cultivar and another on length.In SSR, the example of allelic variation can be: allelotrope A:GAGAGAGA (4 repetitions of GA sequence) and allelotrope B:GAGAGAGAGAGA (6 repetitions of GA sequence).Variation on these length is easily in laboratory, to find, and their allow in the procedure of breeding tracking to genotypic variation.
Term " micro-satellite " is the substituting term of SSR.
Term " single nucleotide polymorphism " or " SNP " be single core thuja acid in the genome (or other total sequence)- a, t, cor g-the mutant dna sequence that occurs when (or between karyomit(e) of individual pairing) between the member of species is different.For example,, from two sections DNA fragmentations that are sequenced of Different Individual, AAGCCTA and AAGCTTA, the difference that comprises the single core thuja acid.We have said two allelotrope: C and T in this case.Almost all general SNP only has two allelotrope.
Term " host cell " refers to the nucleic acid (for example carrier) that comprises allos and the cell that copies and/or express of supporting this nucleic acid.Host cell can be for example intestinal bacteria (E.coli) of prokaryotic cell prokaryocyte, or eukaryotic cell for example yeast, insect, Amphibians or mammalian cell.Host cell can be monocotyledons or dicotyledons cell.The dicotyledons host cell can be that for example, host cell is drawn in Kano.
Term " transgenic plant " refers to the plant that comprises heterologous polynucleotide in its genome.Heterologous polynucleotide generally stably is integrated in genome, makes these polynucleotide be passed to the continuous generation.Heterologous polynucleotide can be individually or is integrated in genome as the part of recombinant expression cassettes." transgenosis " used herein comprises because the heterologous nucleic acids existence has changed genotypic any cell, clone, callus, tissue, plant part or plant, comprises the transgenosis of initially carrying out this type of change and pass through those genetically modified organisms or the cell of hybridizing or vegetative propagation produces from initial genetically modified organism or cell.As used herein, the genome (chromogene group or karyomit(e) alia gene group) that term " transgenosis " is not contained by conventional plant breeding method (that is, hybridization) or by infecting such as random cross fertilization, non-recombinant virus, non-recombinant bacteria transforms, the naturally-occurring event non-restructuring swivel base or spontaneous mutation causes changes.
Term " dicotyledons " refers to the angiospermous subclass that is also referred to as " Dicotyledoneae ", and comprises and refer to whole plant, plant organ (for example, leaf, stem, root, etc.), seed, vegetable cell and its filial generation.As used herein, vegetable cell includes but not limited to seed, suspension culture, embryo, meristematic zone, callus, leaf, root, bud, gametophyte, sporophyte, pollen and sporule.
Term in the context of the invention " hybridization " or " hybridization " refer to produce via pollination the gametogamy of filial generation (that is, cell, seed or plant).This term comprises sexual hybridization (a strain plant is pollinated by another strain plant) and selfing (self-pollination, that is, when pollen and ovule during from identical plant).
Term " infiltration " refers to that the expectation allelotrope of locus is delivered to the phenomenon of another kind of genetic background from a kind of genetic background.For example, desired allelotrope can be passed at least one progeny plant by the sexual hybridization between two stock plants in the infiltration of specific gene seat, and wherein at least one stock plant has desired allelotrope in its genome.Alternatively, for example allelic transmission can occur by the restructuring between two donor gene groups, and for example, in merging protoplastis, wherein one of them donor protoplastis has the allelotrope of expectation in its genome.Desired allelotrope can be, for example, and the selected allelotrope of transgenosis or mark or QTL.
Term " extreme disease pressure field research condition " refer to as, for example, the controlled disease study condition described in WO2006/135717.For example, the petal that extreme disease pressure can be grown surely by application Semen Sesami Nigrum (Niger) seed carrier simulation Sclerotinia produces.The bacterium that makes preparations for sowing after natural kind bacterium can be used as is present in large Tanaka.The per-cent disease incidence of tested plant is adjusted according to running check, and has given 1 to 9 SSDIS scoring.Yet, at these under extreme condition, plant is at least susceptible more due to underlying cause to Sclerotinia: (1) under extreme disease pressure field research condition, plant has stood to irrigate by atomization the humidity provided, this is conducive to the development of Sclerotinia, (2) under extreme disease pressure field research condition, due to artificial covering, plant is in semi-enclosed environment, this has guaranteed to be conducive to the lasting wet condition of Sclerotinia development, and (3) are under extreme disease pressure field research condition, the different tested plant of six row is arranged in each block, therefore the tested plant of any a line that there is the specific modality phenotype may be had the different plant of two row of different morphology phenotypes around.Therefore, for example, from any beneficial effect that less is conducive to the morphology phenotype (high branch) that Sclerotinia infects, be lowered because the plant that any a line may be had a different morphology phenotype (for example, low branch) around.On the other hand, growing plants under natural field condition: (1) is not closed among the artificial covering of having guaranteed lasting humidity, (2) the plant with same modality phenotype around block in grow, this makes the whole beneficial effects from this form to be expressed.Therefore, there is the selection that less is conducive to the form (for example, high branch) that Sclerotinia infects, under natural field condition than carrying out significantly better under extreme disease pressure field research condition.
Very definite, take year is unusual as basis produces reliable land for growing field crops data.Sclerotinia is strong disease, but it only develops in the moist summer of temperature gentleness.For many years, when the condition of Sclerotinia be suboptimum the time, for the screening to the Sclerotinia resistance large Tanaka, a plurality of problems become most important.The existence of the existence of moist time length, water quality, kind bacterium and moist or damp and hot microenvironment affects the development of disease in crop.Although the method that the present invention describes for be Btassica, should be understood that, described method can be applicable to infect any plant of susceptible by thecasporous Sclerotinia.This comprises Sunflower Receptacle (maize ear rot), safflower (maize ear rot), dry beans (pod rot), dry pea (pod rot), soybean (stem rot and pod rot), clover (blossom blight) and lettuce (Lettuce Drop).Bardin and Huang 2001.But the also participate public announcement of a patent application 2003/0150016 of impact about Sclerotinia on soybean.
Large Tanaka's key issue has been solved as follows:
(a) for suitable people's work post bacterium of the continuum of data gathering: because natural kind bacterium always is not triggered large Tanaka, developed simulation by the kind bacterium of the infection of petal.The carrier of fungi can be the Semen Sesami Nigrum seed (little certain herbaceous plants with big flowers (Guizotia abyssinica)-Niger's seed) of surely having grown Sclerotinia, and scatters when integral petal comes off.
(b) water quality and Sclerotinia: initial, underground water is used to irrigate the land for growing field crops that Sclerotinia is grown surely.Yet, in thering are low rainfall and high or low temperature several years, observed and infected lacking of shifting.Vitro test has confirmed that underground water suppresses the growth of Sclerotinia.Chamber and land for growing field crops test, determined that deionization (D1) water treatment has changed the quality of underground water fully by experiment, to prevent the inhibition that Sclerotinia is grown.Afterwards, DI water is used to irrigate extreme disease pressure field research plot.In theory, treated deionized water and initial underground water difference are that mineral (for example magnesium and calcium (lime)) are lowered and pH is unaffected.Sclerotinia produces oxalic acid (a kind of diffusible toxin) and helps course of infection (US 6,380,461).Calcium can be combined with oxalic acid, produces caoxalate.The removal of calcium is likely in deionized water the qualitative change factor that makes Sclerotinia to grow.Therefore, the low or barren water source of mineral content (for example reduced or removed magnesium and calcium) can be used.
(c) irrigation of handling by the blade humidity sensor: for what realize continuing large Tanaka moistening, only used and to have triggered the blade humidity sensor (Campbell Scientific) of irrigating in humidity during lower than the threshold value set.Optimized irrigation makes disease can develop and strengthen the screening to the disease resistance.Yet excessive irrigation may be disturbed significant assessment.Particularly, the genotypic row of uniqueness very close to research equipment in, the lodging of a line can cause by plant and contacting of plant the propagation of pathogenic agent, and improves the disease incidence on the second genotype.Therefore, for the second, genotypic Sclerotinia resistance scoring may have been underestimated its Potential performance in the colony of homogeneity more.In natural land for growing field crops data test, heavy irrigation can produce Sclerotinia than being generally used for given genotypic more favourable environment by the increase of lodging.Therefore, the performance of above-mentioned test subject may be due to heavy irrigation but distortion.
(d) provide and hide the necessary microenvironment of development of helping maintain disease: in order to make disease in drying, sweltering heat and/or can to develop windy season, can use the covering of net formula.
Above-mentioned new method make it possible to realize disease in check development, phenotype reliable expression and under the Sclerotinia condition of optimizing to the sign of multiple different lines.
mark
The invention provides and follow molecule marker chain in quantitative trait locus (" the QTL ") heredity be associated with whole plant field resistance to Sclerotinia.This type of molecule marker is for identifying and producing anti-Sclerotinia or the dicotyledons that has the resistance of the raising of Sclerotinia is useful, particularly, this type of commercially important dicotyledonous crops as draw Sunflower Receptacle, Kano, clover and soybean.
The gene mapping of hundreds of kind molecule marker has generated the genetic linkage maps that covers about 1400cM (centimorgan), corresponding to 19 Kanos, draws karyomit(e).The part that hereinafter title is " selection that the mark of plant is auxiliary and breeding " provides about the character of molecule marker and the additional details of use.
The exemplary indicia locus be associated with whole plant field resistance to Sclerotinia is positioned to 13 linkage group: N1, N3, N4, N7, N8, N9, N10, N11, N12, N13, N15, N18 and N19 in swede type rape (Brassica napus).These exemplary marker gene seats have been described to comprise between the chromosomal region of the quantitative trait locus (QTL) be associated with the phenotype measurement of the whole plant field resistance of whole plant field resistance to Sclerotinia or raising.For example, the table 5 of this paper and 11 has been listed the mark that is positioned to linkage group N1, N3, N4, N7, N8, N9, N10, N11, N12, N13, N15, N18 and N19.Can design additional primer and the probe corresponding to the fragment of these marks or these marks based on sequence information provided herein.
AG0023 (SEQ ID NO:1); AG0045 (SEQ ID NO:2); AG0047 (SEQ ID NO:3); AG0070 (SEQ ID NO:4); AG0086 (SEQ ID NO:5); AG0093 (SEQ ID NO:6); AG0125 (SEQ ID NO:7); AG0148 (SEQ ID NO:8); AG0171 (SEQ ID NO:9); AG0203 (SEQ ID NO:10); AG0239 (SEQ ID NO:11); AG0243 (SEQ ID NO:12); AG0272 (SEQ ID NO:13); AG0304 (SEQ ID NO:14); AG0323 (SEQ ID NO:15); AG0324 (SEQ ID NO:16); AG0328 (SEQ ID NO:17); AG0359 (SEQ ID NO:18); AG0369 (SEQ ID NO:19); AG0370 (SEQ ID NO:20); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0410 (SEQ ID NO:23); AG0441 (SEQ ID NO:24); AG0477 (SEQ ID NO:25); AG0482 (SEQ ID NO:26); AG0504 (SEQ ID NO:27); AG0510 (SEQ ID NO:28); BG0031 (SEQ ID NO:29); BG0106 (SEQ ID NO:30); BG0111 (SEQ ID NO:31); BG0119 (SEQ ID NO:32); BG0181 (SEQ ID NO:33); BG0228 (SEQ ID NO:34); BG0255 (SEQ ID NO:35); BG0278 (SEQ ID NO:36); BG0295 (SEQ ID NO:37); BG0452 (SEQ ID NO:38); BG0516 (SEQ ID NO:39); BG0647 (SEQ ID NO:40); BG0651 (SEQ ID NO:41); BG0713 (SEQ ID NO:42); BG0864 (SEQ ID NO:43); BG0869 (SEQ ID NO:44); BG0988 (SEQ ID NO:45); BG1062 (SEQ ID NO:46); BG1090 (SEQ ID NO:47); BG1101 (SEQ ID NO:48); BG1123 (SEQ ID NO:49); BG1127 (SEQ ID NO:50); BG1149 (SEQ ID NO:51); BG1182 (SEQ ID NO:52); BG1197 (SEQ ID NO:53); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1244 (SEQ ID NO:56); BG1286 (SEQ ID NO:57); BG1288 (SEQ ID NO:58); BG1321 (SEQ ID NO:59); BG1368 (SEQ ID NO:60); BG1392 (SEQ ID NO:61); BG1442 (SEQ ID NO:62); BG1449 (SEQ ID NO:63); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); CA0105 (SEQ ID NO:66); CA0120 (SEQ ID NO:67); CA0163 (SEQ ID NO:68); CA0221 (SEQ ID NO:69); CA0226 (SEQ ID NO:70); CA0233 (SEQ ID NO:71); CA0328 (SEQ ID NO:72); CA0410 (SEQ ID NO:73); CA0423 (SEQ ID NO:74); CA0456 (SEQ ID NO:75); CA0488 (SEQ ID NO:76); CA0546 (SEQ ID NO:77); CA0552 (SEQ ID NO:78); CA0603 (SEQ ID NO:79); CA0614 (SEQ ID NO:80); CA0636 (SEQ ID NO:81); CA0681 (SEQ ID NO:82); CA0719 (SEQ ID NO:83); CA0736 (SEQ ID NO:84); CA0739 (SEQ ID NO:85); CA0753 (SEQ ID NO:86); CA0834 (SEQ ID NO:87); CA0837 (SEQ ID NO:88); CA0896 (SEQ ID NO:89); CA0991 (SEQ ID NO:90); CA1027 (SEQ ID NO:91); CA1032 (SEQ ID NO:92); CA1034 (SEQ ID NO:93); CA1035 (SEQ ID NO:94); CA1066 (SEQ ID NO:95); CA1080 (SEQ ID NO:96); CA1090 (SEQ ID NO:97); CA1097 (SEQ ID NO:98); CA1107 (SEQ ID NO:99); PE0012 (SEQ ID NO:100); PE0017 (SEQ ID NO:101); PE0063 (SEQ ID NO:102); PE0091 (SEQ ID NO:103); PE0131 (SEQ ID NO:104); PE0133 (SEQ ID NO:105); PE0177 (SEQ ID NO:106); PE0187 (SEQ ID NO:107); PE0203 (SEQ ID NO:108); PE0250 (SEQ ID NO:109); PE0281 (SEQ ID NO:110); PE0283 (SEQ ID NO:111); PE0286 (SEQ ID NO:112); PE0324 (SEQ ID NO:113); PE0340 (SEQ ID NO:114); PE0355 (SEQ ID NO:115); UB0015 (SEQ ID NO:116); UB0126 (SEQ ID NO:117); UB0163 (SEQ ID NO:118); UB0181 (SEQ ID NO:119); UB0196 (SEQID NO:120); UB0307 (SEQ ID NO:121); UB0315 (SEQ ID NO:122); UB0331 (SEQ ID NO:123); KK66 (SEQ ID NO:124); And KK98G (SEQ ID NO:125) (sometimes being called as " the illustrated mark of SEQ ID NO:1-125 ").Comprise simple repeated sequence (SSR) polymorphism or single nucleotide polymorphism (SNP), they have been identified the QTL of the whole plant field resistance that contributes to Sclerotinia whole plant field resistance or raising and can be used as its mark.The quantity that should be appreciated that the repetition in SSR can change.For example, in table 7 and table 13, provide and contributed to the whole plant field resistance of Sclerotinia or to the favorable allels of the whole plant field resistance of the raising of Sclerotinia.
It is to be noted, regardless of their molecular property, for example, no matter mark is SSR, AFLP, RFLP etc., and mark is strain specificity normally.That is, specific polymorphism mark, for example exemplary indicia of the present invention mentioned above, be to stipulate with respect to paid close attention to parental line.For each marker gene seat, every a pair of parental line has been identified to allelotrope that resistance is associated and that be associated for susceptibility on the contrary.After the susceptibility by specific allelotrope and the parent of crossbred and resistance are associated, described mark can be used to identify to have the genotypic filial generation corresponding to desired resistant phenotype.In some cases, that is, in the crossbred of some parental lines, exemplary indicia as herein described will not be the information that provides best.In such cases, the additional mark that quantity of information is arranged, for example, and the mark that some is chain and/or homology mark, evaluated and substitute for gene type, such as the selection auxiliary for mark etc.In the situation that mark is corresponding to QTL, after the allelic evaluation that resistance and susceptibility are associated, the direct colony of screening sample likely, the sample for example obtained from seed bank, and need not at first parent's phenotype be associated with allelotrope.
linked marker
Person of skill in the art will appreciate that, in the interval at the mark by mentioned above to restriction, can identify additional molecule marker.This type of mark be also be accredited as the QTL heredity be associated with Sclerotinia whole plant field resistance herein on chain, and be within the scope of the invention.Mark can be identified by any multiple heredity or physical mapping technology.Determine that it is known to those skilled in the art marking whether to follow method chain in QTL (or the specific mark) heredity be associated with resistance to Sclerotinia, and comprise, for example, Interval Mapping (Lander and Botstein (1989) genetics121:185), return graphing method (Haley and Knott (1992) heredity69:315) or MQM graphing method (Jansen (1994) genetics138:871).In addition, the physical mapping technology such as chromosome walking, contig mapping and assembling etc., can be used to identify and separate as the additional sequence that is marked with use in context of the present invention.
homologous nucleotide sequence
In addition, AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; And CA1107; And PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; The homologous nucleotide sequence that has practicality in the evaluation of the QTL be associated for the Sclerotinia whole plant field resistance of identifying in dicotyledons strain, kind or the species from different with KK98G is useful.This type of homology mark is also feature of the present invention.
This type of homologous sequence can be identified by the selective cross with reference sequences.Reference sequences normally derives from unique sequences or its complementary sequence of the listed any marker gene seat of this paper, for example fragment (for example,, corresponding to the AFLP mark) of unique Oligonucleolide primers sequence, EST, amplification etc.
Two kinds of single-chain nucleic acids are when forming duplex " hybridization ".Double-stranded region can comprise one of single-chain nucleic acid or whole both total lengths, or a kind of subsequence of whole and another kind of single-chain nucleic acid of single-chain nucleic acid, or described double-stranded region can comprise the subsequence of each nucleic acid.The nucleic acid that the selective cross condition has been distinguished is relevant to reference sequences (or its complementary sequence) (for example, total significant sequence identity) and in nonspecific mode, with reference sequences, be associated those.In general, the selective cross condition is following those conditions: in pH7.0 to 8.3 salt concn lower than about 1.5M sodium ion, common approximately 0.01 to 1.0M Na ion concentration (or other salt), and for example, be at least about 30 ℃ for short probe (10 to 50 Nucleotide) temperature, and be at least about 60 ℃ for long probe (for example, more than 50 Nucleotide) temperature.The selective cross condition also can realize by adding the destabilizing agent such as methane amide.Selectivity can be hybridized and/or the severity of wash conditions realizes by change.Exemplary low stringency condition is included in 37 ℃ hybridizes in the damping fluid that contains 30 to 35% methane amides, 1M NaCl, 1%SDS (sodium lauryl sulphate), and at 50 to 55 ℃ with 1 * wash to 2 * SSC (20 * SSC=3.0M NaCl/0.3M trisodium citrate).Exemplary medium stringent condition is included in 37 ℃ of in 40 to 45% methane amides, 1M NaCl, 1%SDS hybridizes, and under 55 to 60 ℃ with 0.5 * wash to 1 * SSC.Exemplary high stringent condition is included in 37 ℃ hybridizes in 50% methane amide, 1M NaCl, 1%SDS, and washs with 0.1 * SSC at 60 to 65 ℃.
Specificity depends on post-hybridization washing usually, and the ionic strength of last washing soln and temperature are key factors.Usually, under definite ionic strength and pH, stringent condition is chosen as the pyrolysis chain temperature (T than particular sequence and complementary sequence thereof m) low approximately 5 ℃.Yet utmost point stringent condition can adopt than melting temperature (Tm) (T m) hybridization and/or washing at the temperature of low 1,2,3 or 4 ℃; Medium stringent condition can adopt than melting temperature (Tm) (T m) hybridization and/or washing at the temperature of low 6,7,8,9 or 10 ℃; Low stringency condition can adopt than melting temperature (Tm) (T m) hang down at 11,12,13,14,15 or 20 ℃ of temperature and hybridize and/or washing.
T mthe temperature when complementary target sequence of finger (under definite ionic strength and pH) 50% and the probe hybridization mated fully.For the DNA-DNA crossbred, Tm can enough Meinkoth and Wahl ((1984) anal.Biochem.equation estimation 138:267-284): T m=81.5 ℃+16.6 (%GC)-0.61, (log M)+0.41 (%form)-500/L; The molarity that wherein M is univalent cation, the per-cent that %GC is guanosine and cytidylic acid(CMP) in DNA, the per-cent that %form is methane amide in hybridization solution, and the L length that is the crossbred that means with base pair.The mispairing of every appearance 1%, T mreduce approximately 1 ℃; Therefore, can regulate T mhybridization and/or wash conditions are with the sequence hybridization with having expectation identity.For example, if seek the sequence of have>=90% identity, can be by T mreduce by 10 ℃.
Utilize above-mentioned equation, hybridization and cleaning composition and required T m, those skilled in the art will be understood that, the variation of the severity of hybridization and/or washing soln is described in itself.If required mispairing degree causes T mlower than 45 ℃ (aqueous solution) or 32 ℃ (formamide soln), preferably increase the concentration of SSC in order to can use higher temperature.Hybridization and/or wash conditions can be carried out at least 10,30,60,90,120 or 240 minutes.Detailed guidance for nucleic acid hybridization can be found in Publication about Document: Tijssen (1993) laboratory Techniques in Biochemistry and Molecular? biology--Hybridization with Nucleic Acid Probespart i, the 2nd chapter " Overview of principles of hybridization and the strategy of nucleic acid probe assays " Elsevier, New York.The precaution relevant with damping fluid and incubation conditions etc. to the selection of the hybridization of nucleic acid, probe are discussed, and the general text of multiple other theme of paying close attention in context of the present invention (for example,, corresponding to the use of the order-checking of mark/QTL of the clone of the nucleic acid of mark and QTL, clone, promotor, carrier etc.) can see Berger and Kimmel (1987) guide to? molecular Cloning Techniques, Methods in Enzymologythe 152nd volume, Academic Press, Inc., San Diego (" Berger "); The people such as Sambrook, (2001) molecular? cloning-A Laboratory Manual, the 3rd edition 1-3 volume, Cold Spring Harbor Laboratory, Cold Spring Harbor (" Sambrook "); With people (editor) (supplemented through 2001) such as Ausubel current Protocols in Molecular Biology, John Wiley and Sons, Inc., (" Ausubel ").
Except hybridizing method mentioned above, the homologue of mark of the present invention can also be used any multiple sequence alignment and compare rules and identify by computer.For the purpose of follow-up discussion, following term is used to the descriptive markup nucleotide sequence and with reference to the sequence relation between polynucleotide sequence:
" reference sequences " is the sequence for example be used as, with the restriction on the sequence of cycle tests of the present invention (, candidate's mark homologue) basis relatively.Reference sequences can be the subsequence of specified sequence or all; For example, a fragment of full-length cDNA or gene order, or full-length cDNA or gene order.
As used herein, " comparison window " for example is, by the continuous and specific fragment (, subsequence) with reference sequences polynucleotide/peptide sequence relatively.With regard to the optimized comparison of two kinds of sequences, with respect to one or more interpolations of reference sequences or deletion (the fragment of described polynucleotide/peptide sequence in comparison window can comprise, breach), described reference sequences (according to definition) does not comprise interpolation or deletes.The optimized comparison of two kinds of sequences produces the different Nucleotide/amino-acid residue of minimum number in comparison window.In general, the length of comparison window is at least 20 continuous Nucleotide/amino-acid residues, can be optionally 30,40,50,100 kind of Nucleotide, or longer.Those skilled in the art understands, for fear of owing to comprising wrong high similarity between two kinds of sequences that room causes in polynucleotide/peptide sequence, usually carries out the assessment of gap penalty, and deduct gap penalty from matching number.
" sequence identity " in context in two kinds of nucleic acid or peptide sequence or " identity " refer to compares to seek maximum to identical residue in seasonable two kinds of sequences on specific comparison window.
" per-cent sequence identity " refers to the definite values of sequence of relatively aliging to two kinds of optimizations by comparison window.Per-cent is calculated by the following method: determine that two sequences have the number in the site of identical Nucleotide or amino-acid residue, determine the number in coupling site, the number in this coupling site, divided by the sum in site in comparison window, and is multiplied by 100 per-cents that obtain sequence identity by acquired results.
Have realized that when sequence identity per-cent during for protein, the difference in different residue sites usually is conservative aminoacid replacement, wherein amino-acid residue is substituted by have similar chemical property other amino-acid residue of (for example electric charge or hydrophobicity), and does not therefore change the functional performance of this molecule.When sequence occurs that conserved residues replaces difference, can raise sequence identity per-cent to carry out the correction for the conservative character replaced.Be called as and there is " sequence similarity " or " similarity " by the conservative sequence difference caused that replaces of this class.For the method for carrying out this adjusting, be well-known to those skilled in the art.Usually this relates to and conservatively replaces scoring for part rather than mispairing fully to one, therefore improves sequence identity per-cent.Therefore for example one of them same amino acid must be divided into 1, and non-conservatively replaces to such an extent that be divided into zero, and the conservative score that replaces is between zero-sum 1.According to for example Meyers and Miller (1988) computer Applic.Biol.Sci.the algorithm of 4:11-17 calculates the conservative scoring replaced, for example, and as applied in program PC/GENE (Intelligenetics, Mountain View, California, USA).
Aligned sequences is well known in the art for method relatively.Can be by local homology's algorithm ((1981) of Smith and Waterman for optimized sequence alignment relatively adv. appl.Math.2:482); The homology alignment algorithm of Needleman and Wunsch ((1970) J. mol.Biol.48:443); The similarity searching algorithm ((1988) of Pearson and Lipman proc. natl.Acad.Sci.USA85:2444); And the computerized application of these algorithms carries out, described computerized application includes but not limited to: the CLUSTAL in PC/Gene program (Intelligenetics, Mountain View, California); GAP, BESTFIT, BLAST, FASTA and TFASTA in Wisconsin Genetics Software Package (Genetics Computer Group (GCG), Madison, Wisconsin, USA); The CLUSTAL program is specified in Higgins and Sharp ((1988) gene73:237-244); Higgins and Sharp ((1989) cABIOS5:151-153); The people such as Corpet ((1988) nucleic Acids research16:10881-90); The people such as Huang ((1992) computer Applications in? the Biosciences8:155-65) and the people ((1994) such as Pearson methods in? molecular Biology24:307-331).
The program that can be used to the BLAST family of Database Similarity retrieval comprises: for the BLASTN for RiboaptDB sequence retrieval nucleotide query sequence; For the BLASTX for Protein Data Bank sequence retrieval nucleotide query sequence; For the BLASTP for Protein Data Bank sequence retrieval protein search sequence; For the TBLASTN for RiboaptDB sequence retrieval protein search sequence; With the TBLASTX for for RiboaptDB sequence retrieval nucleotide query sequence.Referring to, for example, current Protocols in Molecular Biology, the 19th chapter, the people such as Ausubel edit, (1995) Greene Publishing and Wiley-Interscience, New York; The people such as Altschul, 1990, j.Mol.Biol.215:403-410; With people (1997) such as Altschul nucleic Acids Res.25:3389-3402.
For the software that carries out the BLAST analysis, can openly obtain, for example,, by the U.S. state-run biotechnology information center (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/).This algorithm comprises that at first being tested and appraised the short word that in search sequence, length is W saves to identify that high Grading sequence is to (HSP), described short word joint when with database sequence in during the byte-aligned of equal length, or the coupling, or meet some on the occasion of threshold score T.T is called as the adjacent byte score threshold.These initial adjacent byte are hit as seed and are retrieved for starting, to find the longer HSP that comprises them.Then, hitting on both direction of these bytes extended along each sequence, until the comparison mark of accumulation can not increase again.With regard to nucleotide sequence, operation parameter M is (to the award mark of a pair of coupling residue; Always>0) and N (to the point penalty of mispairing residue; Always<0) calculate the mark of accumulation.With regard to aminoacid sequence, rating matrix is used to calculate the mark of accumulation.Byte is hit the extension made progress the each party and stopped when following situations occurring: the maximum that the comparison mark of accumulation reaches from it reduces numerical value X; Due to the residue alignment of one or more negative value scorings, the mark of accumulation reaches zero or lower; Or reach the end of one of sequence.The parameter W of BLAST algorithm, T and X have determined sensitivity and the speed of comparison.BLASTN program (for nucleotide sequence) acquiescence adopts 11 word length (W), 10 expected value (E), 100 cutoff threshold (cutoff), M=5, N=-4, and two chains are all compared.With regard to aminoacid sequence, the word length (W) of BLASTP program acquiescence employing 3,10 expected value (E) and BLOSUM62 rating matrix (referring to, for example, Henikoff and Henikoff (1989) proc.Natl. acad.Sci.USA89:10915).
Except calculating per-cent sequence identity, the BLAST algorithm also carry out similarity between two kinds of sequences statistical analysis (referring to, for example, Karlin and Altschul (1993) proc.Nat ' l. acad.Sci.USA90:5873-5877).The measurement of a kind of similarity that the BLAST algorithm provides is minimum and probability (P (N)), and it provides between two kinds of Nucleotide or aminoacid sequence the indication of the probability of coupling occurs by accident.
BLAST retrieval putative protein mass-energy is enough is turned to stochastic sequence by pattern.Yet the protein of many reality comprises nonrandom sequence area, it can be that same aggressiveness bundle (homopolymeric tract), short period repeat or be rich in one or more amino acid whose zones.May align in the zone of this type of low complex degree between incoherent protein, even other zone of protein is fully different.Multiple low complex degree filter can be used to reduce the alignment of this type of low complex degree.For example, SEG (Wooten and Federhen (1993) comput.Chem.17:149-163) and XNU (Claverie and States (1993) comput.Chem.17:191-201) the low complex degree strainer can be used individually or in combination.
Except as otherwise noted, Nucleotide provided herein and protein identity/similarity value is used GAP (GCG version 10) to calculate under default value.
GAP (overall comparison program) can also be used to polynucleotide of the present invention or polypeptide and reference sequences are compared.GAP is used the algorithm ((1970) of Needleman and Wunsch j.Mol. biol.48:443-453), to find two comparison results that maximize the coupling number and minimize the complete sequence of room number.GAP considers all possible comparison and site, room, and prepares the coupling base of maximum number and minimum room.It allows to mate base unit provides room to form point penalty and room extension point penalty.Each formation room, room that GAP must insert it forms point penalty coupling number.If select room extension point penalty to be greater than zero, GAP must form the gap penalty to the intubating length of the room number of times of each room extension point penalty in addition.The room of giving tacit consent to for the Wisconsin Genetics Software Package version 10 of protein sequence forms the point penalty value and room extension point penalty value is respectively 8 and 2.It is 50 that the acquiescence room of nucleotide sequence forms point penalty, and extension point penalty in acquiescence room is 3.Room forms point penalty and room extension point penalty can be expressed as the integer that is selected from 0 to 100.Therefore, for example, room forms and room extension point penalty can be independently of one another: 0,1,2,3,4,5,6,7,8,9,10,15,20,30,40,50,60 or higher.
GAP provides one of them the best member in comparison family.In this family, a lot of members may be arranged, but other member do not have better quality.GAP has shown four quality factor for comparison: quality, ratio, identity and similarity.Quality is that the maximum of aligned sequences is measured.Ratio is that quality is divided by the base number than in the short-movie section.Identity per-cent is the per-cent of the symbol of actual match.The per-cent similarity is the per-cent of similarity sign.Ignore the symbol from the opposite, room.When the score matrix value of pair of symbols is more than or equal to 0.50, (similarity threshold value), give a mark to similarity.The score matrix used in Wisconsin Genetics Software Package version 10 be BLOSUM62 (referring to, for example, Henikoff and Henikoff (1989) proc.Natl.Acad. sci.USA89:10915).
The multiple ratio of sequence is to being used CLUSTAL comparison method (Higgins and Sharp (1989) cABIOS.5:151-153), adopt default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10) to carry out.The default parameters that uses the CLUSTAL method to carry out comparison in pairs is KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
For example, with the per-cent sequence identity of source marking and its reference marker (, any mark as herein described) normally at least 70%, and, upwards get smallest positive integral and can be expressed as the integer be selected between 70 and 99.Therefore, for example, with the per-cent sequence identity of reference sequences can be at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99%.Sequence identity can be used, and for example, BLAST, CLUSTALW or GAP algorithm calculate under implied terms.
the detection of marker gene seat
Corresponding to the mark of the genetic polymorphism between kind of group members can by this area definite several different methods detected (for example, variable sequence, self-sustained sequence replication, simple repeated sequence (SSR), single nucleotide polymorphism (SNP) or the amplified fragment length polymorphism (AFLP) of the amplification of restriction fragment length polymorphism, isozyme mark, allele-specific hybridization (ASH), Plant Genome).
Most of genetic markers rely on one or more characteristics of nucleic acid to be detected.For example, some technology that detect for genetic marker are utilized the hybridization of probe nucleic acid and nucleic acid corresponding to genetic marker.Crossing system includes but not limited to that solution phase, solid phase, mixed phase or in situ hybridization detect.The mark of restriction fragment length polymorphism (RFLP), by will be normally by the probe of the sub-fragment of detected nucleic acid the synthetic oligonucleotide of sub-fragment (or corresponding to), hybridized and be detected with the genomic dna through restrictive diges-tion.Restriction Enzyme is selected as being provided in Different Individual at least two kinds of substituting (or polymorphism) length and the restriction fragment through being everlasting and changing between strain and strain.Determining that generation is simple process for (one or more) Restriction Enzyme of the fragment that information is provided of each hybridization, is well-known in the art.Label probe for example, in suitable matrix (, agarose), according to length, separate and transfer to film (for example, nitrocotton, nylon) upper after, it under the condition that causes the combination of probe balance with target hybridization, remove too much probe by washing subsequently.
Can clone and/or synthesize the nucleic acid probe for the mark seat.The detectable that is applicable to using together with nucleic acid probe comprises by the detectable any composition of spectrum, radio isotope, photochemistry, biological chemistry, immunochemistry, electricity, optics or chemical process.Available marker comprises the vitamin H for dyeing, the markd Streptavidin of its tool, magnetic bead, fluorescence dye, radio-labeled thing, enzyme and colourity mark.Other marker comprises the part with antibodies, and this antibody is by fluorophore, chemical illuminating reagent and enzyme labelling.Making mark be marked with thing can easily realize, for example, by the marker gene seat being used to the PCR primer of tape label thing.
Then the most typically use radioautograph or other similar detection technique (for example, photofulorography, liquid scintillation counter, etc.) to detect the probe of hybridization.The example of specific cross rules is that this area is widely available, referring to for example Berger, Sambrook, Ausubel, all referring to above.
The variable sequence of amplification refers to the extension increasing sequence of Plant Genome, and this sequence shows high nucleic acid residue mutability between the member of same species.All biologies have variable gene group sequence, and every kind of biology (except the clone) has one group of different variable sequence.Once confirm, can use the particular variable sequence prediction phenotypic character of existence.From the effect of preferably playing amplification template together with the primer of the DNA of plant and side joint DNA variable sequence.The amplification variable sequence, then checked order.
Amplification Technologies is well known in this area.Be enough to this type of in vitro method of guidance technology librarian use, (for example comprise the technology of polymerase chain reaction (PCR), ligase chain reaction (LCR), Q β-replicative enzyme amplification and the mediation of other RNA polymerase, the example of technology NASBA), see people's ((1987) United States Patent (USP)s 4 such as Berger, Sambrook and Ausubel (all referring to above) and Mullis, 683,202); pCR Protocols, A Guide to Methods and? applications(CA (1990) (Innis) for (people such as Innis edits) Academic Press Inc., San Diego Academic Press Inc.San Diego); Arnheim and Levinson ((October 1 nineteen ninety) c& EN36-47); the Journal Of NIH Research(1991) 3,81-94; The people such as Kwoh ((1989) proc.Natl.Acad.Sci.USA 86, 1173); The people such as Guatelli ((1990 ) Proc.Natl.Acad.Sci.USA87,1874); The people such as Lomell ((1989) j.Clin.Chem.35,1826); The people such as Landegren ((1988) science241,1077-1080); Van Brunt ((1990) biotechnology8,291-294); Wu and Wallace ((1989) gene4,560); The people such as Barringer ((1990) gene89,117) and Sooknanan and Malek ((1995) biotechnology13:563-564).The improved method of nucleic acid of clone's amplification in vitro is described in the people such as Wallace, and United States Patent (USP) 5,426, in 039.By pcr amplification, the improved method summary of large nucleic acid is in the people such as Cheng (1994) naturein 369:684 and reference wherein, produced therein pcr amplification of maximum 40kb.The technician will know use reversed transcriptive enzyme and polysaccharase, basically any RNA can be changed into to the double-stranded DNA that is suitable for restrictive diges-tion, pcr amplification and order-checking.Referring to Ausubel, Sambrook and Berger, all referring to above.
For (for example,, in amplified reaction) as the oligonucleotide of primer and for use as the oligonucleotide of nucleotide sequence probe usually according to Beaucage and Caruthers ((1981) tetrahedron Lett.the solid phase phosphoramidite three ester methods of 22:1859) describing are chemically synthetic, or can with commodity basis, order simply.
Alternatively, can use autonomously replicating sequence to identify genetic marker.Self-sustained sequence replication refers to use target nucleic acid sequence to carry out a kind of method of nucleic acid amplification, this sequence is carried out external index and is copied by using reverse transcription to copy the following three kinds of enzymic activitys that relate under the condition of isothermal basically: (1) reversed transcriptive enzyme, (2) RNA enzyme H, and (3) rely on the RNA polymerase (people (1990) such as Guatelli of DNA proc.Natl.Acad.Sci.USA87:1874).By cDNA intermediate mimic rna reverse transcription replicanism, this method is gathered cDNA and the RNA copy of primary target.
Amplified fragment length polymorphism (AFLP) also can be used as the genetic marker (people (1995) such as Vos nucl.Acids Res.23:4407.Phrase " amplified fragment length polymorphism " refers to the restriction fragment of the selection of increasing before or after with the restriction endonuclease cracking.Amplification step makes the specific limited fragment detect and is easier to.AFLP allows the detection to a large amount of polymorphism marks, and has been used to the gene mapping (people (1995) such as Becker of plant mol.Gen.Genet.249:65; With people (1995) such as Meksem mol.Gen.Genet.249:74.
Can use allele-specific hybridization (ASH) to identify genetic marker of the present invention.The stabilizing annealing of the fully-complementary sequence of the single strand oligonucleotide probes of ASH technology based on short and strand target nucleic acid.Via the isotropic substance or the heterotope mark that are attached on probe, detected.
With regard to every kind of polymorphism, design two or more different ASH probes, described probe has identical DNA sequence dna except polymorphic Nucleotide.Each probe and an allelotrope sequence will have definite homology so that the scope of probe can be identified all known alternative allelotrope sequences.Each probe and target DNA hybridization.Use suitable probe design and hybridization conditions, the single base mismatch between probe and target DNA will stop hybridization.In this way, only one of them selective probe will with target sample hybridization, described sample be with allelotrope, isozygoty or homogeneity.Two allelotrope be heterozygosis or heterogeneous sample will with two selective probe hybridizations.
Use the ASH mark as the dominant marker, only wherein whether have an allelotrope by a probe hybridization only or do not hybridize to determine.Infer and obtain in the allelotrope that alternative allelotrope can never be hybridized.ASH probe and target molecule are optionally RNA or DNA; Target molecule is the Nucleotide of any length, except the sequence with the probe complementation; Arbitrary chain hybridization of designing probe and DNA target; Confirm the probe size scope, different stringent hybridization conditions etc.
PCR makes the target sequence of ASH increase from the low concentration nucleic acid of relative a small amount of.This external application restriction endonuclease digestion is from the target sequence of genomic dna and separate different big or small sequences by gel electrophoresis.Along with target sequence is attached on the film surface, or, as United States Patent (USP) 5,468,613 described ASH probe sequences can be attached on film, usually hybridize.
In one embodiment, the ASH data obtain by following steps: use PCR amplification of nucleic acid fragment (amplicon) from genomic dna, the amplicon target DNA is transferred on film with the dot blot form, by the oligonucleotide probe of tape label thing and the hybridization of amplicon target, and by the autoradiography observation hybridization spot.
The mark that single nucleotide polymorphism (SNP) is comprised of consensus sequence, this sequence is based on mononucleotide and difference.Usually, the different migration models of the amplicon that this difference can be by comprising SNP on acrylamide gel for example are detected.Yet, do not get rid of substituting detection method, for example, as hybridization, ASH or rflp analysis.
For the another kind of ultimate principle of genetic linkage maps is provided, simple repeated sequence (SSR) utilized in genome high-caliber two, three, four, five or the Hexanucleotide series connection repeat.Dinucleotides repeat to be in the news with from 10 to 60 or more diverse n value the (people (1991) such as Jacob occurs nearly 50,000 times human genome cell67:213.Dinucleotides repeats also to be found (Condit and Hubbell (1991) in higher plant genome34:66).
In brief, by making primer and the Plant Genome conservative region at SSR sequence flank, hybridize, produced the SSR data.Then, the Nucleotide that PCR is used between amplimer repeats.Then, the sequence of amplification is carried out to electrophoresis, to determine its size, thereby and determine two, three and the number of tetranucleotide repeat.The number repeated makes favorable allels and unfavorable allelotrope distinguish.For example, in table 7 and table 13, provide for the whole plant field resistance to Sclerotinia or to the favourable allelotrope of whole plant field resistance of the raising of Sclerotinia.
Alternatively, isozyme mark is used as genetic marker.Isozyme is various form of enzyme, and their amino acid differs from one another, so their nucleotide sequence difference.Some isozyme are the polymer enzymes that comprise slightly different subunits.Other isozyme is polymer or monomer, but in the different loci of aminoacid sequence by cracking from proenzyme.Isozyme can be characterized and be analyzed on protein level, or congenerous different on nucleic acid level can be determined.Can use in these cases any methods analyst isozyme mark based on nucleic acid as herein described.
In an alternative embodiment, the method by computer can be used to the certification mark locus.For example, the nucleotide sequence that comprises described mark can be stored in computer.Desired marker gene seat sequence or its homologue can be used suitable nucleic acid searching algorithm identified, described algorithm as by, for example, such as the program be easy to get of BLAST, provide.
the OTL mapping
Developed the kinds of experiments normal form, to identify and to analyze QTL.In general, these normal forms relate to hybridization one or more pairs of parents, the parent to being, for example, derive from a pair of of two inbred lines, or the multiple relevant or incoherent parent of different inbred lines or strain, it shows the different qualities be associated with paid close attention to phenotypic character separately.Population to parent and filial generation carries out gene type, usually for a plurality of marker gene seats, and with regard to paid close attention to proterties, is assessed.In the context of the present invention, for illustrated one or more molecule markers of any this paper or homologue or the substituting mark chain with illustrated one or more marks of any this paper, parent and progeny plant are carried out to gene type, and just the whole plant field resistance of Sclerotinia or the whole plant field resistance of raising are assessed.Significant statistic correlation between the marker genetype of the progeny plant based on assessed and resistant phenotype, identified the QTL be associated with Sclerotinia whole plant field resistance.Determine that it is known to those skilled in the art marking whether to follow several different methods chain in QTL (or other mark) heredity be associated with whole plant field resistance to Sclerotinia, and comprise, for example, Interval Mapping (Lander and Botstein (1989) Genetics 121:185), recurrence graphing method (Haley and Knott (1992) Heredity 69:315) or MQM graphing method (Jansen (1994) Genetics 138:871).In addition, following application provides the additional details of the substituting statistical method about can be used for complicated breeding population, described method can be used to the people's such as QTL:Beavis that evaluation and location be associated with Sclerotinia whole plant field resistance USSN 09/216, the people's such as 089 " QTL MAPPING IN PLANT BREEDING POPULATIONS " and Jansen PCT/US00/34971 " MQM MAPPING USING HAPLOTYPED PUTATIVE QTLS ALLELES:A SIMPLE APPROACH FOR MAPPING QTLS IN PLANT BREEDING POPULATIONS.”
marker assisted selection and plant breeding
In crop, the major impetus of exploitation molecule marker is to raise the efficiency in plant breeding by marker assisted selection (MAS).Genetic marker allelotrope, perhaps alternatively, certified QTL allelotrope, be used to identify at one or more locus comprise desired genotype and expection by desired genotype and the progeny plant that is passed to them together with desired phenotype.Genetic marker allelotrope (or QTL allelotrope) can be used to identify at a locus or several not chain or linked gene seats and comprise expectation genotypic plant (for example haplotype), and this plant expection is by desired genotype and the filial generation that is passed to them together with desired phenotype.The invention provides to be tested and appraised and (for example there is specific allelotrope, the illustrated mark of one or more this paper or at this paper other mark in listed interval) plant, identify resistance or show the plant to the whole plant field resistance of the raising of Sclerotinia, especially the method for dicotyledons (for example, Btassica).Similarly, be tested and appraised the allelic plant of the expectation that lacks described mark, the plant that can identify susceptible, and, for example, it is got rid of from follow-up hybridization.Should be appreciated that with regard to the purpose of MAS, terms tag can not only contain mark but also contain the QTL locus, because the two can both be used to identify anti-Sclerotinia or have the plant to the resistance of the raising of Sclerotinia.
In desired phenotype (for example, Sclerotinia whole plant field resistance) and the polymorphism chromosomal loci (for example, marker gene seat or QTL) be confirmed as being divided into from after, likely utilize these polymorphic locuses to select the allelotrope corresponding to desired phenotype---be called as the method for marker assisted selection (MAS).In brief, detect from the biological sample of the plant that will be selected corresponding to the nucleic acid of labeling nucleic acid.This detection can be taked the form of probe nucleic acid and mark hybridization, for example, uses the pcr amplification in allele-specific hybridization, southern blotting technique analysis, rna blot analysis, in situ hybridization, the laggard row labels of primer hybridization zone etc.This paper describes the several different methods of certification mark, is for example the part of " detection of marker gene seat " at exercise question.After confirming there is (or not existing) specific markers in biological sample, plant is selected, that is, be used to prepare progeny plant by selection breeding.
The plant breeder need to combine disease tolerance locus and gene for high yield and other desired proterties, to develop improved plant variety.Disease screening to a large amount of samples can be expensive, consuming time and insecure.Using the genetic marker of nucleic acid chain in polymorphic locus as herein described and heredity as disease resistant gene seat, is to select the effective ways of tolerance kind in the procedure of breeding.For example, with regard to the disease resistance, marker assisted selection (MAS) is that MAS can carry out any time in 1 year, is not subject to the restriction of the season of growth with respect to an advantage of field evaluation.In addition, environmental effect and marker assisted selection are irrelevant.
When a population for example, at a plurality of locus that separate the one or more proterties of impact when (relate to a plurality of locus of single resistance of planting disease or each are related to a plurality of locus to the resistance of different diseases), MAS efficiency is compared with phenotypic screen and is become even higher, because all locus can be processed in laboratory together from the single sample of DNA.In example of the present invention, this means and be selected from AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; CA1107; PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; With KK98G or with the multiple mark of their homologies or chain mark, can in the colony of single sample or sample, side by side or sequentially be detected.Therefore, these marks of any one or more, for example, two or more, (and comprising) whole these definite marks at the most, can side by side be detected.In some cases, wish the mark of assessment corresponding to each linkage group be associated with Sclerotinia whole plant field resistance.
MAS another purposes in plant breeding is to assist to recover the recurrent parent genotype by back cross breeding.Back cross breeding is the method that one of filial generation and its parent are backcrossed.In the genetic background that is generally used for infiltrating from one of the donor parent or some locus from other expectation of recurrent parent that backcrosses.The round of carrying out of backcrossing is more, and recurrent parent is larger to the Genetic Contributions of the kind of gained.This is usually essential, because tolerant plants otherwise may be unexpected, that is, because low yield, low setting percentage etc.In contrast, as the kind of the result of the high-intensity procedure of breeding, may there are excellent output, setting percentage etc., for example just lack a kind of desired proterties, as the resistance to special pathogen (, Sclerotinia whole plant field resistance).
By any above listed method, such as RFLP, AFLP, SSR etc., determine the existence in showing the Plant Genome of preferred phenotypic character of specific genetic marker locus or its homologue and/or do not exist.If the nucleic acid from described plant is positive with regard to desired genetic marker, described plant can carry out selfing and has the breeding true strain of homologous genes type with generation, perhaps its can with the plant hybridization that there is same tag or there is other desired characteristic, to produce the hybrid generation of bisexual hybridition.
As described above, the technician will know, QTL described herein has represented the genome area of the gene that includes the Sclerotinia whole plant field resistance that helps plant.In addition, each QTL can differently contribute to this resistance level.Therefore, breeding work has pointed to the quantity that improves these QTL that exist in germplasm, especially quantitatively important QTL.The procedure of breeding early stage, may have less QTL in specific germplasm, but this quantity will improve along with the carrying out of the procedure of breeding.Therefore, in certain embodiments, the plant that shows Sclerotinia whole plant field resistance can comprise at least 6 QTL as herein described.More specifically, described plant can comprise at least 7,8,9 or 10 QTL as herein described.More specifically, described plant can comprise 11,12 or whole QTL as herein described.
positional cloning
Molecule marker of the present invention and with the nucleic acid of its homology, can be used to as mentioned before identify other chain marker gene seat, it can be cloned by improving the flow process of setting up, for example, and as described in detail in Ausubel, Berger and Sambrook (referring to above).Similarly, the chain molecule marker of exemplary indicia and any other evaluation, can be used to separate physically the nucleic acid that (for example,, by the clone) is associated with the QTL that contributes to Sclerotinia whole plant field resistance.This type of nucleic acid (that is, chain with QTL) serves many purposes, and is included in the application of marker assisted selection (MAS) subsequently as genetic marker for the identification of additional QTL.
These nucleic acid are at first identified by the genetic linkage of they and mark of the present invention.The separation of the nucleic acid of paying close attention to realizes by a lot of methods, as at for example Ausubel, Berger and Sambrook, and referring to above, and Clark, editor (1997) plant Molecular Biology:A Laboratory manualspringer-Verlag, discuss in detail in the reference of Berlin.
For example, the gene location clone utilizes the propinquity of genetic marker to limit physically the chromosome segment that separate chain with QTL.The chromosome segment that the method preparation that can know by this type of separates: as with one or more Restriction Enzymes, digested chromosomal DNA, or by polymerase chain reaction (PCR) or substituting amplified reaction amplification chromosomal region.Usually be connected to and be applicable to copying and optionally for example express, in the carrier (, plasmid, cosmid, phage, artificial chromosome etc.) of institute's Insert Fragment through the fragment digested or increase.The mark of contiguous associated with phenotypic character open reading frame (ORF) can be hybridized with DNA clone, thereby identifies the clone that ORF is located thereon.If marking path is far away, by continuous some screening and separating clones of taking turns, identify the fragment that comprises open reading frame, it comprises continuous DNA sequence dna jointly---" contig ".Be enough to the rules that the guidance technology personnel separate the clone associated with linked marker and see, for example Berger, Sambrook and Ausubel, all referring to above.
between the chromosomal region of the nucleic acid of contiguous mark/separation
The invention provides the nucleic acid separated that comprises the QTL be associated with Sclerotinia whole plant field resistance.Described QTL is close to mark as herein described and/or is positioned in the interval limited by two marks of the present invention, and wherein each mark is positioned at the flank of described QTL.This type of nucleic acid and/or interval can be used to identify the nucleic acid of homology and/or can be used to produce to there are the transgenic plant that pass through the field resistance of the whole plant to Sclerotinia that imported QTL gives.Separated between described nucleic acid and/or the chromosomal region that comprises QTL, for example, by positional cloning method mentioned above, be cloned.Can comprise one or more ORF that are associated with resistance between chromosomal region, and can be cloned on one or more independent carriers, for example, according to the size between described chromosomal region.
Should be appreciated that in this area and have variety carrier can be used for the separation of nucleic acid of the present invention and copy.For example, plasmid, cosmid and phage vector are well known in this area, and enough for many application (for example,, for relating to from being less than 1 to the approximately application of the insertion of the nucleic acid of 20 kilobase (kb) scope).In some applications, preparation or clone large nucleic acid, identify chain with given mark further nucleic acid or over the nucleic acid of 10-20kb (for example separate, reach hundreds of kilobase or more, as and the chain mark of chain two of the QTL that identifies as this paper between whole interval, nearly and comprise one or more centimorgans (cM)), be favourable.In this case, in this area, have the multiple carrier that can hold large nucleic acids to use, these comprise yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), plant artificial chromosome (PAC) etc.To the generality introduction of the YAC as artificial chromosome, BAC, PAC and MAC referring to, for example, Monaco and Larin (1994) trends Biotechnol.12:280.In addition, for the method for amplification in vitro and the chain large nucleic acids of genetic marker, be available (for example, the people (1994) such as Cheng at large nature369:684, and reference wherein).Clone's system can be fabricated or commercialization ground obtains; Referring to, for example, Stratagene Cloning Systems, catalogue 2000 (La Jolla, CA).
the generation of transgenic plant and cell
The invention still further relates to and use host cell and the organism transformed corresponding to the nucleic acid of identifying QTL and other gene according to the present invention.For example, this type of nucleic acid comprises between chromosomal region, ORF and/or cDNA, or corresponding to being included between identified chromosomal region or the sequence in ORF or subsequence.In addition, the invention provides polypeptide corresponding to QTL by the production of recombinant technology.With the present invention's carrier of the present invention (host cell is, comprise QTL or other nucleic acid and carrier as described above that the method according to this invention is identified) genetically engineered (that is, transduction, transfection or conversion), described carrier comprises, for example, cloning vector or expression vector.Except above-described those, examples of such carriers also comprises, for example, and the polynucleotide of Agrobacterium (agrobacterium), virus (for example plant virus), " naked " polynucleotide or coupling.Carrier is imported into vegetable cell or the plant protoplast of plant tissue, cultivation by the multiple standards method, described method comprises the electroporation (people (1985) such as From proc.Natl.Acad.Sci.USA 82; 5824), virus vector infects the (people (1982) such as Hohn as cauliflower mosaic virus (CaMV) molecular Biology of Plant Tumors(Academic Press, New York, 549-560 page); Howell United States Patent (USP) 4,407,956), be used in microballoon or particle matrix or surface on there is nucleic acid particulate carry out high speed and bombard and penetrate (the people (1987) such as Klein nature327; 70), make carrier (WO85/01856) with pollen or with the agrobacterium tumefaciens that is carried at the T-DNA plasmid of wherein having cloned DNA fragmentation (Agrobacterium tumefaciens) or Agrobacterium rhizogenes (A.rhizogenes).The T-DNA plasmid is passed in vegetable cell by agrobacterium tumefaciens infection, and a part of T-DNA plasmid is advanced the (people (1984) such as Horsch in Plant Genome by stable integration science233; 496; The people such as Fraley (1983) proc.Natl.Acad.Sci.USA80; 4803).The method that nucleic acid of the present invention is imported to host cell is also non-key for the present invention.Therefore, can use any method of nucleic acid to the effective importing in cell or protoplastis is provided, for example, include but not limited to example above.
Can be in the metabolism substratum culturing engineering host cell, described substratum through suitable modification to obtain this type of active as activation promotor or to be selected from transformant.Optionally cultivate these cells, make them enter transgenic plant.From the protoplastis of cultivating, aftergrowth is described in the people such as Evans ((1983) " Protoplast Isolation and Culture, " handbook of Plant Cell? cultures1,124-176 (MacMillan Publishing Co., New York); Davey ((1983) " Recent Developments in the Culture and Regeneration of Plant Protoplasts, " protoplasts, 12-29 page, (Birkhauser, Basel)); Dale ((1983) " Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops, " protoplasts, 31-41 page, (Birkhauser, Basel)); And Binding ((1985) " Regeneration of Plants, " plant Protoplasts, 21-73 page, (CRC Press, Boca Raton)).
The invention still further relates to for example, generation with the genetically modified organism of nucleic acid (, clone's QTL of the present invention) transduction, described genetically modified organism can be bacterium, yeast, fungi or plant.Discussing in detail of the technology relevant with bacterium, unicellular eukaryote and cell culture is found in the reference of above enumerating and outlines hereinafter.The several well-known process that target nucleic acid is imported to bacterial cell is available, and the present invention can use any method.These comprise: make recipient cell and the bacterium protoplast fusion that comprises DNA, carry and with viral vector infection (being specified in hereinafter) etc. with the liposome-treated cell that comprises DNA, electroporation, projectile body bombardment (particle gun), carbon fiber.Bacterial cell can be used to the quantity of plasmid that amplification comprises DNA construct of the present invention.Bacterial growth is to logarithmic phase, and the plasmid in bacterium can separate by several different methods known in the art (referring to, for example, Sambrook).For purifying, from the plurality of reagents box of the plasmid of bacterium, be commercially available acquisition in addition.According to the specification sheets of manufacturers correctly use they (referring to, for example, EasyPrep tM, FlexiPrep tM, they are all from Pharmacia Biotech; StrataClean tM, from Stratagene; And QIAprep tM, from Qiagen).Then through the plasmid of separation and purifying, to prepare other plasmid, described plasmid is for transfection of plant cells or be integrated into the agrobacterium tumefaciens related vector with infection plant in further manipulation.General carrier comprises transcribes with translation termination, transcribes and translate initiating sequence and for regulating and controlling the promotor of particular target expression of nucleic acid.Carrier optionally comprises the genetic expression box, and this expression cassette comprises at least one independently terminator sequence, the sequence (for example shuttle vectors) that allows this box all to copy in eukaryote or prokaryotic organism or two kinds of cells and for the selective marker of prokaryotic system and eukaryotic system.Carrier is suitable for prokaryotic organism, eukaryote or preferably in both, copies and integrate.Referring to, Giliman and Smith ((1979) gene8:81); The people such as Roberts ((1987) nature328:731); (the people (1995) such as Schneider protein Expr.Purif. 6435:10); Ausubel, Sambrook, Berger (all referring to above).By for example American type culture collection (ATCC), provided for clone's bacterium and the catalogue of phage, for example, the aTCC Catalogue of Bacteria and Bacteriophage(1992) people (editor) such as Gherna, ATCC publishes.About the additional basic procedure of order-checking, clone and molecular biological other side, and consider people such as being found in Watson (1992) as basis theoretic recombinant DNA, second edition, Scientific American Books, NY.
nucleic acid is proceeded to plant
Embodiments of the invention relate to the production of the transgenic plant of the nucleic acid that comprises clone of the present invention (for example, between the chromosomal region, be associated with QTL, separate ORF and cDNA).For being common obtainable by the technology of nucleic acid transformed plant cell, and can encoding or, corresponding to the nucleic acid between the chromosomal region of QTL, QTL homologue, separation etc., be applicable to the present invention by use.Except Berger, Ausubel and Sambrook, the useful general reference of cloning, cultivating and regenerate about vegetable cell comprises Jones (editor) ((1995) plant Gene Transfer and? expression Protocols--Methods in Molecular Biology, the 49th volumehumana Press Towata NJ); The people such as Payne ((1992) plant Cell and Tissue Culture in Liquid systemsjohn Wiley & Sons, Inc.New York, NY (Payne)); With Gamborg and Phillips (editor) ((1995) plant Cell, Tissue and Organ Culture; fundamental Methodsspringer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) is (Gamborg)).The various kinds of cell culture medium be described in Atlas and Parks (editor) ( the Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL (Atlas)) in.The document that the Additional Information of culture plant cell is found in commercially available acquisition as life Science Research Cell Culture Catalogue(1998), derive from Sigma-Aldrich, Inc. (St Louis, MO) (Sigma-LSRCCC), and, for example, plant Culture Catalogueand supplement (1997), derive from Sigma-Aldrich equally, and Inc. (St Louis, MO) (Sigma-PCCS).Additional detail about culture plant cell is found in Croy, (editor) ((1993) plant Molecular Biologybios Scientific Publishers, Oxford, U.K.).
By multiple routine techniques, nucleic acid construct of the present invention is imported to the vegetable cell in culture or plant organ as plasmid, cosmids, artificial chromosome, DNA and RNA polynucleotide.Wherein express described sequence, this sequence optionally is combined with transcribing and translate the startup regulating and controlling sequence, and described regulating and controlling sequence instructs pirate recordings or the translation from the sequence of foreign DNA in the destination organization of conversion of plant.
Can the nucleic acid of separation of the present invention be imported to plant according to multiple technologies known in the art.Be also well known for the technology that transforms multiple higher plant species, and be described in technology, science and patent documentation.Referring to, for example, the people such as Weising (1988) ann.Rev.Genet.22:421-477.
DNA construct of the present invention, for example, the DNA polynucleotide of plasmid, phagemid, cosmid, phage, naked polynucleotide or multiple coupling (for example, the DNA of the DNA of the DNA of polylysine coupling, polypeptide coupling, liposome coupling etc.) or artificial chromosome, can utilize the technology such as electroporation and microinjection plant protoplast, directly imported the genomic dna of vegetable cell, perhaps described DNA construct can utilize the particle gun method as the DNA partickle bombardment, is directly imported vegetable cell.
For the injection, for example, cell, plumule, callus and protoplastis, microinjection technique, be known in the art and be specified in science and patent documentation in.For example, several different methods is described in Jones (editor) ((1995) plant Gene Transfer and Expression Protocols-- methods in Molecular Biology, the 49th volumehumana Press Towata NJ), and in other reference of mentioning of this paper, and be found in document.
For example, use polyethylene glycol precipitation to import DNA construct, be described in the people such as Paszkowski (EMBO is (1984) J.3:2717).Electroporation technology be described in the people such as Fromm ( proc. nat ' l.Acad.Sci.USA82:5824 (1985)).The via Particle Bombardment Transformation technical description in the people such as Klein ( nature327:70-73 (1987)).Additional details sees Jones (1995) and Gamborg and Phillips (1995), referring to above, and in United States Patent (USP) 5,990,387.
Alternatively, the conversion of Agrobacterium mediation is used to produce transgenic plant.The transformation technology of Agrobacterium mediation, comprise removing toxic substances and the use of binary vector, also is specified in scientific literature.Referring to, for example, the people such as Horsch (1984) science233:496; With people (1984) such as Fraley proc.Nat ' l.Acad.Sci.USA80:4803, and summarize in Hansen and Chilton (1998) current Topics in Microbiology240:22 and Das (1998) subcellular Biochemistry 29:Plant Microbe Interactionsin the 343-363 page.
DNA construct can and be imported into conventional agrobacterium tumefaciens host carrier with the regional combination of suitable T-DNA side joint.When the agrobacterium tumefaciens infection cell, agrobacterium tumefaciens host's pathogenic function will guide construct and contiguous mark to insert plant cell dna.Referring to United States Patent (USP) 5,591,616.Although Agrobacterium is mainly used in dicotyledons, some monocotyledons can be transformed by Agrobacterium.For example, the Agrobacterium of corn transforms and to be described in United States Patent (USP) 5,550, in 318.
Other method of transfection or conversion comprise (1) Agrobacterium rhizogenes mediation conversion (referring to, for example, Lichtenstein and Fuller (1987): genetic Engineering, the 6th volume, PWJ Rigby, editor, London, Academic Press; And Lichtenstein; And Draper (1985) C.P.: dNA Cloning, the II volume, D.M.Glover edits, Oxford, IRI Press); WO 88/02405, be published on April 7th, 1988, described agrobacterium rhizogene strain A4 and its use of Ri plasmid together with agrobacterium tumefaciens carrier pARC8 or pARC16, (2) liposome-mediated DNA take in (referring to, for example, the people (1984) such as Freeman plant Cell physiol.25:1353), (3) vortex method (referring to, for example, Kindle (1990) proc. natl.Acad.Sci., (USA)87:1228).
DNA can also proceed to and be imported into plant by direct DNA in pollen, as the people such as Zhou ((1983) methods in Enzymology, 101:433); Hess ((1987) intern Rev. cytol.107:367); With people ((1988) such as Luo plant Mol.Biol.Reporter6:165) described.Can be by DNA being injected to the expression that the plant generative organ obtains the peptide coding gene, as the people such as Pena ((1987) nature325:274) described.Also DNA directly can be injected to the dry embryo cell of prematurity plumule and rehydration, as the people such as Neuhaus ((1987) theor.Appl. genet.75:30); With people such as Benbrook, ((1986) exist proceedings Bio Expobutterworth, Stoneham, Mass., 27-54 page) described.The various plants virus that can be used as carrier is known in the art and comprises cauliflower mosaic virus (CaMV), geminivirus infection, bromovirus and tobacco mosaic virus (TMV).
the regeneration of transgenic plant
Can cultivate the transformed plant cells obtained by any above-mentioned transformation technology and there is the genotype of conversion the whole plant that therefore there is desired phenotype with regeneration.This type of regeneration techniques relies on the certain plants hormone of handling in the tissue culture growth medium, usually relies on biocide and/or the weedicide mark be imported into together with desired nucleotide sequence.From the protoplastis of cultivating, aftergrowth is described in the people such as Evans ((1983) Protoplasts Isolation and Culture, Handbook of Plant Cell culture, 124-176 page, Macmillian Publishing Company, New? york); And Binding ((1985) Regeneration of Plants, Plant Protoplaststhe 21-73 page, CRC Press, Boca Raton).Also can be from plant callus, explant, somatocyte plumule (people (1989) the J.Tissue Cult. such as Dandekar meth.12:145; The people such as McGranahan (1990) plant Cell Rep.8:512) organ or their part realize regeneration.This type of regeneration techniques is described in the people ((1987) such as Klee in general manner ann.Rev.of Plant Phys.38:467-486).Additional details sees Payne (1992) and Jones (1995), and all referring to above, and Weissbach and Weissbach edit, ((1988) methods for Plant? molecular Biologyacademic Press, Inc., San Diego, CA).This regeneration and process of growth comprise the following steps: select the cell and the seedling that transform, make the transformant seedling rooting, and make the plantlet growth in soil.Make these methods be suitable for the present invention, with preparation, carry the QTL of the method according to this invention separation and the transgenic plant of other gene.
In addition, comprise the regeneration of plant that is imported into the polynucleotide of the present invention of leaf explant cell by Agrobacterium, can be as the people such as Horsch ((1985) science227:1229-1231) described realization.In the method, under the existence of transformant selective agent, grow in the substratum of inducing the plant species seedling regeneration be converted, as the people such as Fraley ((1983) proc.Natl.Acad.Sci. (U.S.A.)80:4803) described.The method generates seedling two usually to surrounding, then these transformant seedlings is transferred in suitable seedling inducing culture, and this substratum comprises selective agent and prevents the microbiotic of bacterial growth.Transgenic plant of the present invention can be reproducible or irreproducible.
For transforming and expressing the QTL associated to the whole plant field resistance of Sclerotinia and identify according to the present invention and the plant of other nucleic acid of clone includes but not limited to, important species on agronomy and on Horticulture.These type of species mainly comprise dicotyledons, for example, following section: Cruciferae (Brassicaceae), pulse family (Leguminosae) (comprising pea, beans, Lens culinaris, peanut, yam bean, cowpea, lamb's-quarters beans, soybean, trifolium, clover, lupine, vetch, lotus, Melilotus suaveolens Ledeb., Chinese wistaria and sweet pea); And, composite family (in vascular plant, the section of maximum, comprise at least 1,000 genus, comprises that important cash crop are as Sunflower Receptacle).
In addition, for the target with nucleic acid transformation of the present invention, and above indicate those, plant from following genus: allium (Allium), apium (Apium), Arachis (Arachis), Btassica (Brassica), Capsicum (Capsicum), olecranon Macroptilium (Cicer), Cucumis (Cucumis), Cucurbita (Curcubita), Daucus (Daucus), Fagopyrum (Fagopyrum), Glycine (Glycine), Helianthus (Helianthus), Lactuca (Lactuca), Lens culinaris belongs to (Lens), tomato belongs to (Lycopersicon), Medicago (Medicago), Pisum (Pisum), Phaseolus (Phaseolus), Solanum (Solanum), Trifolium (Trifolium), Vigna (Vigna), with many other.
The common crop plants that is target of the present invention comprises soybean, Sunflower Receptacle, rape, pea, beans, French beans, peanut, yam bean, cowpea, lamb's-quarters beans, trifolium, clover, lupine, vetch, Melilotus suaveolens Ledeb., sweet pea, field pea, broad bean, Caulis et Folium Brassicae capitatae, brussels sprouts, Caulis et Folium Brassicae capitatae, Cauliflower, kale, black salted turnip, celery, lettuce, Radix Dauci Sativae, onion, capsicum, potato, eggplant and tomato.
In recombinant expression cassettes building process of the present invention, (expression cassette comprises; for example; the helper plasmid that comprises the function of causing a disease and comprise plasmid or the virus of exogenous DNA array as structure gene), the plant promoter fragment is optionally used to instruct nucleic acid to express in any or all of tissue of the plant of regeneration.The example of constitutive promoter comprise cauliflower mosaic virus (CaMV) 35S transcribe original area, derive from agrobacterium tumefaciens T-DNA 1 '-or 2 '-promotor and transcribe original area from other of various plants known to the skilled.Alternatively, plant promoter can guide the expression (tissue-specific promoter) of polynucleotide of the present invention in particular organization, or otherwise can under more accurate environment control condition, guide the expression (inducible promoter) of polynucleotide of the present invention.Example in grow controlling lower tissue-specific promoter only comprises to be organized at some, for example fruit, seed or the promotor of spending middle startup to transcribe.
Any can being applicable in a plurality of promotors that guiding is transcribed in vegetable cell.This promotor can be constitutive promoter or inducible promoter.Except above-mentioned promotor, the bacterial origin promotor worked in plant also comprises that octopine synthase promoter, nopaline synthase promoter and other derive from the promotor of natural Ti-plasmids.Referring to, the people such as Herrara-Estrella ((1983), nature, 303:209).Viral promotors comprises cauliflower mosaic virus 35S and 19S RNA promotor.Referring to, the people such as Odell ((1985) nature, 313:810).Other plant promoter comprises ribulose-1,5-bisphosphate, 3-bisphosphate carboxylase small subunit promotor and phaseolin promoter.Also can use the promoter sequence from E8 gene and other gene.The separation of E8 promotor and sequence are specified in Deikman and Fischer ((1988) eMBO J.7:3315).Many other promotors of current use, and promotor can be connected on exogenous DNA array with the guiding expression of nucleic acid.
If the expectation express polypeptide, comprise by those of the QTL be associated with phenotypic character of the present invention or other nucleic acid encoding, in 3 of coding region '-the polyadenylation zone of end normally is included.This polyadenylation district can be derived from natural gene, is derived from multiple other plant gene or is derived from for example T-DNA.
Comprise sequence (for example, promotor or coding region) and the genetically modified carrier of the present invention of gene from the coding expression product, by generally including the nucleic acid subsequence, give the mark of the selectable or phenotype that can screen of vegetable cell.For example, described mark codified biocide resistance, especially antibiotics resistance, for example, to the resistance of kantlex, G418, bleomycin, Totomycin, or Herbicid resistant, for example resistance of or glufosinates (activeconstituents of weedicide bialaphos or Basta) grand to chlorine sulphur.Referring to, for example, the people such as Padgette (1996): herbicide-Resistant Crops(Duke edits), 53-84 page, CRC Lewis Publishers, Boca Raton (" Padgette, 1996 ").For example can give the selectivity of crop to particular herbicide in crop by the through engineering approaches gene, described genes encoding is from other biological suitable herbicide metabolism enzyme as microorganism.Referring to Vasil (1996): herbicide-Resistant Crops(Duke edits), 85-91 page, CRC Lewis Publishers, Boca Raton) (" Vasil ", 1996).
After the technician will recognize in recombinant expression cassettes is stably imported to transgenic plant and confirms that it can operate, can it be imported to other plant by sexual hybridization.Depend on the species that will be hybridized, can use any in the multiple standards breeding technique.In asexually propagated crop, ripe transgenic plant can be by transplanting or tissue culture technique breeds to prepare a plurality of identical plants.Select the expectation transgenosis and obtain new variety, its vegetative propagation is used for to business.In the farm crop of seminal propagation, ripe transgenic plant can selfing and produce the self-mating system plant of isozygotying.The selfing plant produces the seed of the heterologous nucleic acids that comprises new importing.These seeds can grow to produce plant, and this plant will produce selected phenotype.The present invention includes the part of obtaining from the plant of regeneration, such as flower, seed, blade, branch, fruit etc., precondition is the cell that these parts comprise the nucleic acid with separation of the present invention.Also within the scope of the invention, precondition is the nucleotide sequence that these parts comprise importing for the filial generation of the plant of regeneration and modification and mutant.
By, for example, the immunoblotting of standard and DNA detection technology, can be for the transmission of nucleic acid of the present invention, and the transgenic plant of polynucleotide of the present invention are expressed in screening.Can at first measure the expression of rna level, to identify and to measure the plant of positive expression.The standard technique that can use RNA to analyze, described technology comprises the pcr amplification detection method of the Oligonucleolide primers that uses the allos RNA template that is designed to only to increase, and the solution hybridization detection method of using the heterologous nucleic acids specific probe.Then, the Western immunoblotting assay of the antibody of reaction specifically of the application of the invention, can be analyzed the plant of the RNA positive with regard to protein expression.In addition, can carry out in situ hybridization and immunocytochemical assay according to standard schedule, this analysis is positioned at the expression sites in genetically modified organism with heterologous nucleic acids specificity polynucleotide probes and antibody respectively.Usually the importing nucleic acid of the many transgenic strains of screening is to identify and to select to have the plant of optimal express spectra.
An embodiment is with regard to the heterologous nucleic acids with regard to adding to be the transgenic plant of isozygotying; That is, the transgenic plant that comprise two sections nucleotide sequences that add, the homologous genes seat on gene each karyomit(e) right at karyomit(e).Comprise single transgenic plant of planting the heterozygosis of the heterologous nucleic acids added, make the seed germination of some generations and for respect to control plant (by sexual mating (selfing), natural, not genetically modified) the resulting plant of expression analysis that changes of polynucleotide of the present invention, can obtain the transgenic plant of isozygotying.Also imagined with mother plant backcross and with the outcrossing of not genetically modified plant.
high flux screening
In one aspect of the invention, the allelic mensuration of genetic marker is undertaken by high flux screening.High flux screening relates to the library that genetic marker is provided, and for example RFLP, AFLP, isozyme, specific alleles and variable sequence, comprise SSR.Then screen this type of library for Plant Genome, to produce each studied plant " fingerprint ".In some cases, in paid close attention to region generating the local fingerprint of subdivision that comprises described mark.After having identified the genetic marker allelotrope of plant, based on method of the present invention, by statistical correlations, determine the correspondence between one or more marker alleles and desired phenotypic character.
High flux screening can carry out in many different forms.Hybridization can be with 96-, 324-or 1524-well format, or occurs in the matrix on silicon or with other form.
In a kind of form commonly used, dot hybridization equipment is used to make the sample deposition of genomic dna of fragmentation and sex change on nylon or nitrocellulose membrane.By be exposed to UV-light or by heat, make nucleic acid and film crosslinked after, hatch described film with the hybridization probe of tape label thing.Marker is impregnated in nucleic acid probe by well known method in any multiple this area.The washing film, to remove the not probe of hybridization, measure the association of marker and target nucleic acid sequence.
Developed multiple well-known robot system for high flux screening, especially with the form in 96 holes.These systems comprise similar to Takeda Chemical Industries, the Automation workstation of the automatic synthesis equipment of LTD. (Osaka, Japan) exploitation, with many robot system (Zymate II that utilize mechanical arm, Zymark Corporation, Hopkinton, MA.; ORCA tM, Beckman Coulter, Fullerton CA).Any aforesaid device all is suitable for the present invention.For character and the enforcement of the adjustment to these equipment that makes these equipment to move as discussed herein (if having any this type of adjust), for those skilled in the relevant art, will be apparent.
In addition, high throughput screening system self be commercially available acquisition (referring to, for example, Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc.Fullerton, CA; Precision Systems, Inc., Natick, MA etc.).The flow process that the common automatization of these systems is whole, comprise whole samples and reagent move the distribution of liquid, liquid, regularly hatch and finally in the detector that is applicable to this detection to the reading of microwell plate or film.These configurable systems provide high-throughput and have started fast and flexibility ratio and customization highly.The manufacturers of this type systematic provides for use the detailed rules of their product in high throughput applications.
In a modification of the present invention, the solid phase array is used to detect fast and specifically multiple polymorphic nucleotide.Usually, nucleic acid probe is connected to solid support, target nucleic acid with probe hybridization.Probe or target or the two can both be by (normally by fluorophor) marks.If target is labeled, by detecting the fluorescence assessment hybridization of combination.If probe is labeled, hybridization is detected the cancellation of marker by the nucleic acid of combination usually.If probe and target all are labeled, the detection of the hybridization gamut that the vicinity of the marker by monitoring two kinds of combinations causes usually carries out.
In one embodiment, the array that has synthesized probe on solid support.By chip concealing technique and light protection chemistry, likely produce the nucleic acid probe array of customization.These are called as, for example, and " DNA chip " or ultra-large immobilized polymer array (VLSIPS tMarray) array can have about 1cm 2to several cm 2the matrix of area on comprise the probe area in 1,000,000 restriction.
In another embodiment, capillary electrophoresis is used to analyze polymorphism.When polymorphism is based on size, for example, AFLP and SSR, the effect of this technology is best.This technology is described in detail in United States Patent (USP) 5,534, in 123 and 5,728,282.In brief, with isolation medium filled capillary pipe electrophoresis pipe.Isolation medium comprises Natvosol, urea and optional methane amide.AFLP or SSR sample to kapillary, and are carried out electrophoresis by loading.Because capillary electrophoresis only needs a small amount of sample and isolation medium, be very short working time.Measure molecular size by the techniques described herein, thereby and measured the quantity of the Nucleotide existed in the nucleic acid samples.In high throughput format, many kapillaries are placed in capillary electrophoresis.Sample is upper to these pipes by loading, and the electrophoresis of sample side by side carries out.Referring to, Mathies and Huang (1992) Nature 359:167.
the system of integrating
Because the possible number of combinations existed in an array is huge, in one aspect of the invention, the system of integrating is as computer, corresponding to the software of statistical model of the present invention and corresponding to the data set of genetic marker and phenotypic number, the mapping that has helped phenotypic character is comprised QTL.In the context of the present invention, phrase " system of integration " refers to a kind of like this system: wherein enter the external treatment of the data of computer corresponding to physical target or computer, for example, nucleic acid array hybridizing and the processing in computer, this processing causes that input signal is changed into different output signals by physics.In other words, the input data, for example, the hybridization of specific region on array, be converted into the output data, for example, the evaluation of the sequence of hybridization.Processing in computer is a set of instruction or " program ", identifies positive hybridization signal and individual sample is classified as to genotype by integration system thus.Additional program, by the genotype of each sample, is more specifically haplotype in the method for the invention, with phenotypic number, is associated, and for example, uses HAPLO-IM of the present invention +, HAPLO-MQM and/or HAPLO-MQM +model.For example, program
Figure BDA00003691347400651
with especially be applicable to such analysis, and can be expanded as comprising HAPLO-IM of the present invention +, HAPLO-MQM and/or HAPLO-MQM +model.In addition, exist multiple, for example, for the C/C++ program calculated, for the Delphi of gui interface and/or java applet with for example, for the Active X application (, Olectra Chart and True WevChart) of drafting instrument.In integration system of the present invention, other useful Software tool comprises that statistical package is as SAS, Genstat and S-Plus.In addition, additional programming language also is applicable to integration system of the present invention as Fortran etc.
In one aspect, the invention provides the integration system that comprises computer or computer-readable medium, described medium comprises and has at least one database corresponding to the data set of the gene type to genetic marker.Described system also comprises user interface, allows the user optionally to consult one or more databases.In addition, can also Application standard text-processing software for example, as Word (Microsoft Word tMor Corel Wordperfect tM) and database or electrical form software (for example, electrical form software is as Microsoft Excel tM, Corel Quattro Pro tM, or database program as Microsoft Access tMor Paradox) for example, handle together character string with user interface (, at standard operation system as the GUI in Windows, Macintosh or linux system).
The present invention also provides for having comprised the integration system of robot device's sample operation as previously described.Mechanical manipulator liquid is for example controlled armature, for solution (vegetable cell extract) is transferred to point of destination from a source, for example from titer plate, transfer to array substrate, optionally, this armature be may be operably coupled to digital machine (or being connected to the additional computer in integration system).Input unit is generally a feature of integration system, and this device shifts to control armature control high-throughput liquid by mechanical manipulator liquid for data being inputted to digital computer, and the transfer of optionally by armature control, arriving solid carrier.
Integration system for genetic marker analysis of the present invention generally includes digital machine, it has one or more high-throughput liquid and controls software, image analysis software, data interpretation software, the mechanical manipulator liquid for solution is transferred to point of destination from a source that may be operably coupled to digital machine is controlled armature, control to pass through mechanical manipulator liquid the input unit (for example computer keyboard) that armature control high-throughput liquid shifts to digital machine for inputting data, and the image reading apparatus from the marker signal of the probe of tape label for digitizing optionally, described probe, for example, with expression product, hybridize may be operably coupled on the solid support of digital machine.The image reading apparatus interface has image analysis software, in order to provide right, for example, the measurement of the different labeled nucleic acid probe intensity when hybridizing from the array sample nucleic acid population, wherein whether the probe mark ionization meter is made an explanation and is hybridized and hybridize to which kind of degree with show tags probe and marker by data interpretation software.Then, use statistical models of the present invention, the data that so produce are associated with phenotypic number, to determine phenotype and to the correspondence between the gene type of genetic marker, thereby determine chromosome position.
Optical imagery, for example (for example pass through photographic camera or other recording unit, photorectifier and data storage equipment) observe the crossing pattern of (and optionally record), optionally further processed in any embodiment of the invention, for example,, by the described image of digitizing and/or store on computers and analyze this image.The peripherals of multiple commercially available acquisition and software can be used for digitizing, storage and analyze digitized video or digitized optical imagery, for example use the PC (DOS based on Intel x86 or pentium chip compatibility tM, OS2 tMwINDOWS tM, WINDOWS NT tMor WINDOWS95 tMmachine), based on MACINTOSH tM, LINUX or UNIX (for example, SUN tMworkstation) computer.
test kit
Also provide test kit to help for label screening germplasm of the present invention.Test kit comprises polynucleotide of the present invention, its fragment or complementary sequence, they as probe or primer for detection of Sclerotinia whole plant field resistance or to the mark of the whole plant field resistance of the raising of Sclerotinia.Use the specification sheets of described polynucleotide and buffer reagent and/or other solution also can be provided, to help to use described polynucleotide.Described test kit, for high flux screening, particularly, is used the high flux screening of integration system, is useful.
example
Following experimental technique provides the additional details about the particular aspects of the rules relevant to enforcement of the present invention and method with result.The example of the present invention that is subject to claims protections for explanation is provided without restriction, and it is known and be specified in the application of the rules in the reference that this paper quotes that these examples relate to those skilled in the art.Example 1-6 has described the analysis of a swede type rape (Brassica napus) mapping population.Example 7-10 has described the analysis of different swede type rape (Brassica napus) mapping population.
example 1: mapping population 1
Parent for described mapping population is 04DHS11418 (a kind of Sclerotinia resistance double haploid, as ATCC accession number PTA-6778 by preservation) and PHI2004HS1 (the DH strain is drawn in a kind of non-resistance Kano that has a polymorphism with respect to resistant strain, is selected because it has similar agronomy phenotype and has therein stable high susceptibility in selecteed test environment because of resistance in the 04DHS11418 strain) (referring to table 2).These strains are used to produce the double haploid mapping population be comprised of 186 filial generations.
table 2: measure the land for growing field crops performance (research trial) under extreme disease pressure
*sSDI%: sclerotinite disease incidence %.SSDI% is that the deviation adjustment UNSSDI for the 04DHS11418 (25%) with expection and PHI2004HS1 (75%) check mean value marks (per-cent that wherein UNSSDI is the plant that infected by Sclerotinia in population), described in table 4.Above-mentioned scoring is only used under controlled extreme disease pressure field research condition.It is that the SSDI% observed by use is multiplied by factor X calculating, and wherein factor X makes the average SSDI% of suitable check become 50% the factor.Do not carry out the adjustment for severity.
*sSDIS: sclerotinite SSDIS is for the spring habit Kano, being pulled in the deviation adjustment UNSSDI scoring of mean value of the check 46A65/46A76 of the expection under extreme disease pressure.It is that the UNSSDI observed by use is multiplied by factor X calculating, and wherein factor X makes the average SSDI% of suitable check become 50% the factor.Carried out the adjustment for severity after the incidence adjustment.
UNSSDS is the scoring of disease development degree on affected plant.Two kinds of standard of measurements have been used in the present invention.It is from 1 (death) to 9 (disease-free) and the common measure scope is from 0 (disease-free) to 5 (death) plant that pioneer SSDS measures scope.About the details of pioneer SSDS standard of measurement, referring to the table 15 of WO 2006/135717.The common measure standard provides as follows: 0=is disease-free; 1=lesion surface or little branch are influenced; The branch death that 2=is large; The main stem at least 50% of 3=is wound around; The main stem of 4=is wound around, but plant produces good seed; The main stem of 5=is wound around, a large amount of underproduction.
Two kinds of parents all have good lodging resistance.Therefore, lodging resistance or lodging tolerance are fixed, thereby have got rid of this variable in the mapping process.The selection of height susceptible strain has caused not having the population (that is, whole resistances are from 04DHS11418, and there is no the DH daughter lines be that specific resistivity parent resistance is higher) of over parent segregation.Through the time of 4 years, above-mentioned population is carried out to phenotype analytical large Tanaka, and carry out gene type with the SSR molecule marker.Phenotype analytical carries out described in WO 2006/135717, and its complete instruction content is incorporated herein by reference.
example 2:RNA express spectra
Carried out that use 04DHS11418, PHI2004HS1, resistance and batch (comprise susceptible with double haploid resistance (DH) filial generation) susceptible the experiment of rna expression spectrum.Used int leaf texture (not inoculation) and with the leaf texture of samplings in 6,24 and 48 hours after mycelium inoculation.Comprise by use chip that Btassica (85,820) and Arabidopsis (Arabidopsis) (17,617) coerce relevant nucleotide sequence and carry out probe in detecting to different batches, carried out the rna expression spectrum analysis.The multiple gene relevant to Sclerotinia disease resistance or with pectin relevant gene (these genes and cell walls integrity are relevant) with after mycelium inoculation 6,24 and 48 hours, locating to raise.The summary of result is found in table 3.In resistance and strain susceptible, some in these genes are checked order, to find any SNP.Then, these SNP are added in gene mapping and (are called as KK).
table 3: the quantity of the disease genes involved raised in different treatment time.
example 3: Sclerotinia screening
the disease scoring
For disease, the plant of the double haploid mapping population of generation described in example 1 is marked, described in table 4.Unadjusted parameter (for example, UNSSDI and UNSSDS) has shown the variation year by year caused as variation and the meteorological conditions of position in land for growing field crops due to the variation of environment.This type of variation will be that those skilled in the art expects.
table 4: Sclerotinia (Sclerotinia) the parameter UNSSDI that collect in land for growing field crops and UNSSDS with and they are (natural/as to study number with derivative parameter S SDI%, SSDIS (data) and SSFS according to) relation.
Figure BDA00003691347400692
Figure BDA00003691347400701
UNSSDI is the per-cent that is subject to the plant of Sclerotinia infection in population.
UNSSDS is the scoring of disease development degree on affected plant.Two kinds of standard of measurements have been used in the present invention.It is from 1 (death) to 9 (disease-free) and the common measure scope is from 0 (disease-free) to 5 (death) plant that pioneer SSDS measures scope.About the details of pioneer SSDS standard of measurement, referring to the table 15 of WO 2006/135717.The common measure standard provides as follows: 0=is disease-free; 1=lesion surface or little branch are influenced; The branch death that 2=is large; The main stem at least 50% of 3=is wound around; The main stem of 4=is wound around, but plant produces good seed; The main stem of 5=is wound around, a large amount of underproduction.
SSDI% is the deviation adjustment UNSSDI scoring for the 04DHS11418 (25%) with expection and PHI2004HS1 (75%) check mean value, described in table 4.Above-mentioned scoring is only used under controlled extreme disease pressure field research condition.It is that the SSDI% observed by use is multiplied by factor X calculating, and wherein factor X makes the average SSDI% of suitable check become 50% the factor.Do not carry out the adjustment for severity.
SSDIS is the deviation adjustment UNSSDI scoring for the 04DHS11418 (25%) with expection and PHI2004HS1 (75%) check mean value, described in table 4.Above-mentioned scoring is only used under controlled extreme disease pressure field research condition.It is that the UNSSDI observed by use is multiplied by factor X calculating, and wherein factor X makes the average SSDI% of suitable check become 50% the factor.Carried out the adjustment for severity after the incidence adjustment.
SSFS is measuring of the disease incidence of large Tanaka under natural disease pressure and severity.Being calculated as follows of it: SSFS=[SSDI% * SSDS (0-5 measures)] ÷ 5
example 4: gene mapping and qtl analysis
Use JoinMap v3.0 (Van Ooijen, J.W. and R.E.Voorrips, 2001
Figure BDA00003691347400711
3.0, Software for the calculation of genetic linkage maps.Plant Research International, Wageningen, the Netherlands) and carried out gene mapping and qtl analysis.Used Kosambi centimorgan function.If the LOD scoring has surpassed 2.0 threshold value, show that it is QTL.
gene mapping
Gene mapping is placed in 19 linkage groups (Lg) by 351 molecule markers, and these linkage groups are drawn karyomit(e) and public linkage group name corresponding to 19 Kanos.Covered～1400cM of linkage map.
qtl analysis
Between the employing simple zones, (Zeng (1994), qtl analysis Genetics136:1457) has been identified 7 linkage groups (N1, N7, N9, N11, N12, N18 and N19) that contribute to the whole plant field resistance of Sclerotinia for mapping and composite interval mapping (CIM).In addition, by the one-way analysis of variance to the Sclerotinia parameter in P≤0.01 significance level (PROC GLM, SAS Enterprise Guide 4.2), confirmed to map and be accredited as the zone be associated with the Sclerotinia resistance by interval.These QTL have been identified in table 5 hereinafter and 6.As the phenotype of the explanation " change " by table 6 as shown in being worth, some QTL have the larger impact on the Sclerotinia resistance than other.
Use JoinMap v3.0 (Van Ooijen, J.W. and R.E.Voorrips, 2001
Figure BDA00003691347400712
3.0, Software for the calculation of genetic linkage maps.Plant Research International, Wageningen, the Netherlands) and carried out gene mapping and qtl analysis.Used Kosambi centimorgan function.If the LOD scoring has surpassed 2.0 threshold value, show that it is QTL.LOD represents the logarithm (take 10 end of as) of probability.
table 5: the mark be associated with Sclerotinia (Sclerotinia) resistance significantly with P≤0.01
Figure BDA00003691347400713
Figure BDA00003691347400721
Figure BDA00003691347400741
table 6: the QTL be associated with Sclerotinia (Sclerotinia) whole plant land for growing field crops tolerance.
Figure BDA00003691347400742
The Additional Information of the SSR mark of seven QTL side joints that are associated about the whole plant field resistance with to Sclerotinia is presented in the table 14 of example 12.Forward and reverse primer sequence for each mark also are provided." repetition " means the SSR or the SNP that are associated with each mark.The position display of SSR and SNP is being arranged in the last sequence information of specification sheets.
The allelotrope of each SSR mark of 7 QTL side joints that are associated about the whole plant field resistance with to Sclerotinia and the Additional Information of allelotrope size are provided in table 7.
table 7: with (SCL) each SSR mark of QTL side joint of seven Sclerotinias (Sclerotinia) allelotrope and the allelotrope size of note.
Figure BDA00003691347400751
Figure BDA00003691347400761
Figure BDA00003691347400771
Figure BDA00003691347400781
Figure BDA00003691347400791
example 5: the checking of 7 QTL that are associated with whole plant field resistance to Sclerotinia
Under extreme disease pressure field research condition, in the spring habit Kano from Pioneer Hi-Bred, draw 16 kinds of Sclerotinia resistances and the breeding product 10 kinds of Sclerotinia susceptibles of plan to fasten, carried out the simplation verification of 7 QTL that the whole plant field resistance with to Sclerotinia is associated.This makes it possible to the extreme disease condition of annual development, regardless of natural environment.
The table 8 vide infra, the SSR mark for seven QTL side joints with identified, carried out gene type to each resistant strain.In table 9, the first digit in the QTL group refers to linkage group, and second digit refers to the QTL numbering on this linkage group.This is marked at the position (in centimorgan) on linkage group the numeral of the token name side of deserving to be called.Each allelotrope is expressed i) only in resistant strain, there is (light gray), ii) only in the susceptible strain, there is (Dark grey), or iii) all there is (white) in resistance and susceptible strain.By each breeding strain is appointed as to the numerical value addition of 1 (allelotrope only existed), 0.5 (allelotrope all existed) or 0 (allelotrope only existed) in the susceptible strain in resistance and susceptible strain in resistant strain, obtained the sum of favorable allels.The per-cent of favorable allels is from the 63-90% scope in resistant strain, and in the breeding strain of susceptible 13-47% only.This dependency shows, with the mark that is accredited as seven QTL side joints that are associated with Sclerotinia in the breeding population, can be used to select have the individuality of the favorable allels of maximum quantity in the breeding population.These individualities with the highest favorable allels per-cent will be selected as the good candidate with regard to the Sclerotinia resistance.
Figure BDA00003691347400811
Figure BDA00003691347400821
example 6: the Sclerotinia resistance is from spring habit swede type rape (Brassica napus) to the winter habit wild cabbage the infiltration of type rape (Brassica napus).
In parents' basis, 3 sides or composite crossing, make Sclerotinia resistance source 04DHS11418 and winter habit Kano draw incross (for example, as shown in table 9).Then the F1 hybrid is backcrossed with new susceptible parent.In BC1F1 generation, produced about 500 filial generations (for the identification of at least one the individual required minimum quantity with whole beneficial genes existence) for each hybridization, and submitted to for adopting and draw in the spring habit Kano labeled analysis that is accredited as the mark be associated with Sclerotinia.Just, from the existence of the favorable allels of Sclerotinia resistant strain 04DHS11418, checked each individual sample.Calculated the per-cent of the favorable allels existed in each sample, be used to backcross with recurrent parent from the highest three of each colony.Again repeated this process in the BC2 stage.In addition, also make the individuality of choosing mutually match, with the individuality developed therein, can be accredited as the allelic colony of the Sclerotinia with the expectation of isozygotying.
table 9: the Sclerotinia of applying marking assisted Selection (Sclerotinia) resistance is to the winter habit cabbage type the example that rape (B.napus) infiltrates
Figure BDA00003691347400831
Figure BDA00003691347400841
example 7: mapping population 2
For the parent of described mapping population, be 06DSB13911 (a kind of Sclerotinia resistance double haploid) and PHI2008HS1 (the DH strain is drawn in a kind of Kano that has a susceptible of polymorphism with respect to resistant strain, because it has similar agronomy phenotype and has therein stable high susceptibility in selecteed test environment because of resistance in the 06DSB13911 strain, is selected) (referring to table 10).These strains are used to produce the double haploid mapping population be comprised of 187 filial generations.
table 10: measure the land for growing field crops performance (research trial) under extreme disease pressure
Figure BDA00003691347400842
*sSDI%: sclerotinite disease incidence %.SSDI% is the deviation adjustment UNSSDI for the 06DSB13911 (15%) with expection and PHI2008HS1 (75%) check mean value mark (per-cent that wherein UNSSDI is the plant that infected by Sclerotinia in population).Above-mentioned scoring is only used under controlled extreme disease pressure field research condition.It is that the SSDI% observed by use is multiplied by factor X calculating, and wherein factor X makes the average SSDI% of suitable check become 45% the factor.
*sSDIS: sclerotinite SSDIS is for the spring habit Kano, being pulled in the deviation adjustment UNSSDI scoring of the check mean value of the expection under extreme disease pressure.Above-mentioned scoring is only used under controlled extreme disease pressure field research condition.It is that the UNSSDI observed by use is multiplied by factor X calculating, and wherein factor X makes the average SSDI% of suitable check become 45% the factor.Carried out the adjustment for severity after the incidence adjustment.
UNSSDS is the scoring of disease development degree on affected plant.Two kinds of standard of measurements have been used in the present invention.It is from 1 (death) to 9 (disease-free) and the common measure scope is from 0 (disease-free) to 5 (death) plant that pioneer SSDS measures scope.About the details of pioneer SSDS standard of measurement, referring to the table 15 of WO 2006/135717.The common measure standard provides as follows: 0=is disease-free; 1=lesion surface or little branch are influenced; The branch death that 2=is large; The main stem at least 50% of 3=is wound around; The main stem of 4=is wound around, but plant produces good seed; The main stem of 5=is wound around, a large amount of underproduction.
Two kinds of parents all have good lodging resistance.Therefore, lodging resistance or lodging tolerance are fixed, thereby have got rid of this variable in the mapping process.The selection of height susceptible strain has caused not having the population (that is, whole resistances are from 06DSB13911, and there is no the DH daughter lines be that specific resistivity parent resistance is higher) of over parent segregation.Through the time of 3 years, above-mentioned population is carried out to phenotype analytical large Tanaka, and carry out gene type with the SSR molecule marker.Phenotype analytical carries out described in WO 2006/135717, and its complete instruction content is incorporated herein by reference.
example 8: Sclerotinia screening
the disease scoring
For disease, the plant of the double haploid mapping population of generation described in example 7 is marked, described in the table 4 of example 3.Unadjusted parameter (for example, UNSSDI and UNSSDS) has shown the variation year by year caused as variation and the meteorological conditions of position in land for growing field crops due to the variation of environment.This type of variation will be that those skilled in the art expects.
example 9: gene mapping and qtl analysis
Use JoinMap v3.0 (Van Ooijen, J.W. and R.E.Voorrips, 2001
Figure BDA00003691347400851
3.0, Software for the calculation of genetic linkage maps.Plant Research International, Wageningen, the Netherlands) and carried out gene mapping and qtl analysis.Used Kosambi centimorgan function.If the LOD scoring has surpassed 2.0 threshold value, show that it is QTL.LOD represents the logarithm (take 10 end of as) of probability.
gene mapping
Gene mapping is placed in 19 linkage groups (Lg) by 278 molecule markers, and these linkage groups are drawn karyomit(e) and public linkage group name corresponding to 19 Kanos.Covered～1100cM of linkage map.
qtl analysis
Between the employing simple zones, (Zeng (1994), qtl analysis Genetics136:1457) has been identified 12 linkage groups (N1, N3, N4, N8, N9, N10, N11, N12, N13, N15, N18 and N19) that contribute to the whole plant field resistance of Sclerotinia for mapping and composite interval mapping (CIM).In addition, by the one-way analysis of variance to the Sclerotinia parameter in P≤0.01 significance level (PROC GLM, SAS Enterprise Guide 4.2), confirmed to map and be accredited as the zone be associated with the Sclerotinia resistance by interval.These QTL have been identified in table 11 hereinafter and 12.As the phenotype of the explanation " change " by table 12 as shown in being worth, some QTL have the larger impact on the Sclerotinia resistance than other.
table 11: the mark be associated with Sclerotinia (Sclerotinia) resistance significantly with P≤0.01
Figure BDA00003691347400861
Figure BDA00003691347400891
Figure BDA00003691347400901
Figure BDA00003691347400911
table 12: with the whole plant land for growing field crops of Sclerotinia (Sclerotinia) whole plant field resistance or raising the phenotype that the QTL of the QTL that resistance is associated is interval, LOD marks and explain changes
Figure BDA00003691347400921
Figure BDA00003691347400931
The Additional Information of the SSR mark of 12 QTL side joints that are associated about the whole plant field resistance with to Sclerotinia is presented in the table 14 of example 12, wherein also provides for the exemplary forward of each SSR and the group of reverse primer sequence." repetition " means the SSR or the SNP that are associated with each mark.In the sequence information of the position display of SSR in example 12.The allelotrope of each SSR mark of 12 QTL side joints that are associated about the whole plant field resistance with to Sclerotinia and the Additional Information of allelotrope size are provided in table 13.
table 13: with each SSR mark of 12 Sclerotinias (Sclerotinia) QTL side joint allelotrope and allelotrope size, and with regard to Sclerotinia (Sclerotinia) (SCL) resistance and say favourable allelotrope.
Figure BDA00003691347400941
Figure BDA00003691347400951
Figure BDA00003691347400971
Figure BDA00003691347400981
Figure BDA00003691347401001
Figure BDA00003691347401011
Figure BDA00003691347401021
Figure BDA00003691347401041
Figure BDA00003691347401051
example 10: the Sclerotinia resistance is sweet to winter habit from spring habit swede type rape (Brassica napus) the infiltration of blue type rape (Brassica napus).
In parents' basis, 3 sides or composite crossing, make Sclerotinia resistance source 06DSB13911 and winter habit Kano draw incross.Then the F1 hybrid is backcrossed with new susceptible parent.In BC1F1 generation, produced about 800-1000 filial generation (for the identification of at least one the individual required minimum quantity with whole beneficial genes existence) for each hybridization, and submitted to for adopting and draw in the spring habit Kano labeled analysis that is accredited as the mark be associated with Sclerotinia.Just, from the existence of the favorable allels of Sclerotinia resistant strain 06DSB13911, checked each individual sample.Calculated the per-cent of the favorable allels existed in each sample, be used to backcross with recurrent parent from the highest three of each colony.Again repeated this process in the BC2 stage.In addition, also make the individuality of choosing mutually match, with the individuality developed therein, can be accredited as the allelic colony of the Sclerotinia with the expectation of isozygotying.
example 11: use the Sclerotinia resistant strain to carry out the production of crossbred seed
With regard to SSDIS, have 5 and the Sclerotinia resistant strain of higher scoring be selected for that the crossbred seed is produced and crossbred is tested.The production of these seeds can be carried out according to known to the skilled and for example method described in WO 2006/135717.
example 12: the flag sequence of Including Polymorphism and exemplary primer
Hereinafter listed the sequence information of the mark of the QTL be associated with Sclerotinia whole plant field resistance significantly with P≤0.01, as shown as example above.In these sequences, the Nucleotide of n=the unknown; Sequence with underscore means the primer sequence from table 14 hereinafter, and the sequence in square brackets means polymorphic regions (SSR, SNP).
AG0023(SEQ?ID?NO：1)
CGAATTCGCCCTTCTCTTGCTTAGATCTGGACTAACTACTTCnnAAAGAAAACATTnnnTTAATGTTTATGTCGAATGTCATTTATGCTGAACAAAATAACCTTGAAAATATGTTCTGTAGGCTAAAGTTGGGAGAGAGAAGGAGGTTGAAGAGATTTTGTCAAGATTGCGAGGAGAAAATTCTGATGTATCAGATGAGGCAGGAGAGATATTAGTAAGCATATATATGCATGAATAATCATATGATCAATGTATATATTTTTTACTTCACAATATTTTGATGATCATCAGGCATATACAGAACATGTTAAACA ACAAGGAGATGATCGCGGTTTCCTCAAGTTGTTTCAGCGAAAATACGCGTTCTCACTTACTGTAATT[CTTCTTCTTCTTCTTCTTCTTCTTTTTCTTCTTCTTCTTCTTCTT]TAATAA CCCGTT TGGTTTACACAGATTGGAGTTGTTCTTATAGCTTTGCCTCAACTTGGAGGTCTTAGTGGTTATTCTTTTTACACTGAGTCCATTTTCATATCTACAGGTAnnnTAACTCTTACTTCTTCAACAAAATCTTGATTTTTATATATTTATTTACCGTAACGATAATTGTTGATAATTACGnnnATCAGGTGTATCGAGTGATGTTGGATTCATATCGACATCTATAGTTC
AG0045(SEQ?ID?NO：2)
ACGAATTCGCCCTTCTCTTGCTTAGATCTGGACTAnnnnnTGATTTGCCCGCTATGTTCGACGGGTGGAGATTTTAGTTTTACTTCCTCGATCTGATTGTATGGGTTGGGAGTAGGGTCTAATATATCAACTGCGAGTGTATGTTCGTTTCCTCCTCAGTTTCGAAGTTGGGTTCTTATGTGTTTAGCCTAAGnnnCTGTGAnnnGnTAGTTTTTTTTTAATCAGTTCCAACAGGATTCATTTCAGGnnnTTGGAACTTGTGTATATGTGTTAGCCTGAGATCTCTGTAGTGTCCGGAAATGATATTTnnnnATTATCATTAATTTAGTTCGAAGnATGAAGCTCAGTGTTGTTGGACTTGTGTATATGGAGCTCGAAGAGTGAAGCTCAGTGCGTTTTCATCTGAGGATGATGATGATGGAGCTAATGTGCTGAGCAATGAGAACTCGAGATG ATAAGGCTTGAGGGACATGCCAGTGAGT[GAAGAAA]CCGTCGGGCTATAGCTTAGT[GAAGAAGAAGAAGAA]GAGCTCGTGGAGTGATCAAATTTGCAGGTATGCCCAAACTTGCCAATCCCACATTGTGGAGAATGGCTGCATTTTTACCA CAAAGCTGTTTCTGTGGAGCCAAAAATGAATGGAGGATAGTAAAACAGAACGTCATAATCAAATCAAGAAATTTTAACTTTTTTTGTCAGCACAAATTTnnnCTTTATCTTTAATTATTTAC
AG0047(SEQ?ID?NO：3)
CGAATTCGCCCTTCTCTTGCTTAnnnnCTGGACTAACAACAATTCCAAAATACTAATTCACAAACTTTGTTTACAATCCAAAGAAAATCCGCTCTTTTGAAGCGCGGATCAAGATCTAGTGTTATAATATATCTAGAACATGGGAGTTTGGTCCAATGAACTACTGTATAGTTCTATCGAAATTTTTGAGTGATAAGATTGAAGCTCCAGCACTCACTTATCTATTTGAGAAGCAAATAATAGAAAAAGAAGTAGATTTGAGGAAGAGATGATGGAGTTGAACAAGGAGCTTTAAGATTTGAGTTCTGACAGTGTA GAAGCTGCAATACTGAGGCAC CAAGGAAGAAAT[CATCATCATCATCATCATCATCATCAT]CAAGAATTAGTTTCAGTTCATATCCACAACCATTTTTTCTTTCAAAGAAATTTGCTGGTAGTAATTTTGAAGTTGTAAATTTTACATTTTCAGTGTTTCATTTTTCTCACGTTTTCTTAATAATTGTTTACTTGCCAAATGATTCCATCACTTGGAAACTCACTATTGTTTGACATTTTGGTGTGCTTAAGTGACTCTTTTCGAGTATTCATACATTATAGAAATTG TTTGGGACAACAGGTAAGAATTGCTTGGCACAAGTAATGGCATCCCTCCCTGCAAATATATATAAATATTACAGTTGTCCTGGAACTTTTnnnnTCTATCCTCTGCTGACAGGATGAGATATATGCATATAGAATATTAACTTCnnTCnGCCCGTATGTTCATGGATGnnnAGCTCCAT
AG0070(SEQ?ID?NO：4)
CGAGGAGTTGAATGACCCTGACTGTACTTTGGCCTCGAGACAGTCCCATCAAGAATAATTTACTGGGTCGATTTTTATTTTTAATTCTGGTCGAGCCAACTCCG AACTGGTCGAGCGGGATTTTTTAATTCTGGTCGACCAAAATCATATCCGCTCGTGAGGGTCTTTACAACCACCATCACCACACTCGGACGATCACCCCACCACCACTTGGACGA[CCACCACCACCACCA]CCGGCGGCTCGGCTAGCTCTCGGGGGGCTCGCGGCGAG[GAGAGA]GGAAGATATCCNACGGAAAGAGAAAAGAGAGG[GAGAGAGAGAGAGA]GGCGTGAGAGAAGAAGAGAGAAAAGGAAAAGAGAA GCTTGACGGCTAGGGTT TCCTAGTCTCTATAAATTCCTGCAGAGCTTCACTCAAGTTTCAGAATGAGAGAAGAGTAAGAGGAGGCAGCTTCATTTATAGAAACAGGAGGAAACCCTAGGTCATTTACCCTAATGGGCTGCAGTCTTAATGGGCTCTCCTTAAGAAAATTTTGGGCTAGGAACCGGGACGTTACAATAATGCTTCTTATGAATATGTCTGAGTAGTTCTTTTGTTAGATTTAGGGTTCTTCAAGGGGTGAATTATGGTTTGCTANATTTATATTGTTGTTTGTGTGATTT
AG0086(SEQ?ID?NO：5)
GCTCGCCGACTTCGGAGTCGCCTCGCCGCTCGATATCCCTTTCAGCCTCGCCTCCATCTCTTTTCCCCAAACTCTAGGCTGTTGCTGTTGCGTCGCCGCCGCCGCCGCCGTGGCTGTTATCGAGCTATTTGATCTACCGTACAGCATTTTAAACCGTTGATCAGATTCGGGATCAGACTTTGTCGTCACCGGAGGGCTCTTGATCGGCGGTTGCACTTCCCCCTCCGTACACGGCGTA C AATGTCGGTAAGCACCGGAAGCTTTCAGAGCCATATCTTTGAGCTGAGAATGAATTTACGAAAATACCCTTGATCAGTATAGAGAATGACAAGAGGTGGAGGATGAGCAAA[GAGAGAGAGAGAGAGAGAGA]AGTCTACCTGAGATGTTAGAGATTTGGCTTGCTTGGAATCCGGATCGTCGGGTTGACCCGAGGTTTCATCGCCGGCTCGCTTCGAACGAGCTA TACAAGTCAGCATT TTCCGGCAGCTGCTGTTTCTTGGTAATGTGATTTTGTTTCTTCTCTTTTTGGATACG[GAGAGA]CAGTAGATGCTGTCAGTTTCTAACTTTGGTTTGTGTTGTGTGTTTGGTCATGGTGCTCTTTTTATGTTTTATACTCACTTTACCANNGAAAACGGTTCCATTTTTTTAA
AG0093(SEQ?ID?NO：6)
TAGGAGATGAGATGTACTGTTGCTTAGGGCTCTTATTTCTCTTGAAACTAGAATAAGCTGCCATCGGGTCGGTGTAATAATCAAACCTTGGCTTATCATACGATTCCTGCTGATGTATGGAT GCTTCAGCCAAGGGATTTGAG AGGTGACTTGTGTTCATAGAGGTTCCAAGCTCTGTAGAACCATCATTCTCTG[CAGCAGCAGCAGCAGCAG]CTTCCATCCGCATTGCTTTTAGCATTT[CTTTTCTTTT]CTCTGAATCTTCCATTACTGCTCAGCTTCAAAGCTAATCAACTACAAAAATATAAACTTTTTTTCGAAATTATCA ATCGAATCGCACC AAAAGAGCTAAGATCTCCACGCGAGAACAATCTAACTAACCCTAAACCCCCAAATTATCCCAAACTCTGTACGGATACTCAAATTGGAAAAGCGAAATTGAGAGGATGCTAACCTTGGTTTACTCAACTTCTTCACTTCCTGGTCGCCAGAGGTAGAGGATGAATGACAAGTGAAAACCCAGAACACGATGATGACGACAACNAAGCCTCCACAAATAAATATAANACCCGGTTCGTGTTCGACCGTGTTTTTCCNATTAAAACCGGTTTACGGCGATNAGAATCATAAACCAAATACGATNATCACGAAGGGTGACGATTAANACGAGACTTCCCAAAACCGGTTCGT
AG0125(SEQ?ID?NO：7)
CTGTTGAGGGGAGGAAACAAGAGCCTGGGAGGAGAACTCCTTGCTGGGGAAGACGAGATCATTCTCCTCAGAGGCAATGGATTCACCTAAGACCACAGCGTTTAACTGAGAGATCTTGCCGGCACCTGAGGGTAGCAGAGACATGGACT CCACATGCCTTAGGTGATTGGATGA?CATTGTCTTACACCGGAGAAGTTTGTCCAACGGAGATGATCTGCCACACCCTTACAAGTCAGATGTCATTTGAATAAAATTTAAAACAAAACCACAAATGTCTTTTTGACTTATTTATCAAAACTGCCTAAACCCCAAACCCAAT[CATCATCATCATCATCATCAT]AACCATATTCATCAATCATTCTATCATTATTGTCATCATTGGATCAGATTATTCATT CACCTTTGAGAAGCCGGAAGAATCCGAGATCCAAGTGATCCGCTTGTTTTCAGATCCTGAAGAAACAAAAACAGATCANAGGCGAATATTCTTTTTTGATTACNATCAGATCATAAGAAGAAGAANAGATTGAAACTTTCGTANACCCAAAACATATCATTATGACNAAAGATCACATCTTTAACTCCNATGATCCCTAAGATTCGACTTACAGGTCGAGAACGAAGAGAGGAAATTTTTTGAAAAATTGTAAGAAGGGGCG
AG0148(SEQ?ID?NO：8)
CTTGAGAGAGAGATTGAAAGTGAGCTGCGCCAAGAACCAAAACGGACATATCACAAGGCTTACTTTAGCAACACG CATCCTTGTCCAACGTCC CTTCTCATCTAAACCTTTAGCACAAATCTCACCACTAACATCAAGCTCTACACTTTCCACACCAGATTCAATCCCACTACCATCTCCCTCCTCTTCTCTAACAACAACCCCTGATCCTTCAGACACATCTGACA[CCACCACCACACCACCTCCTCCT]AGCAAAACATCCTTCTGTGACTCAGCCGACTCCTGCAAATGAGACCTTTTGCTTCTCCGAAACAGTATACTCCCATACGCATCTGCTAAATAC CGACCGATCTCGAAGAGAGGAAGGTTAGTCCAGATCTAAGCAAGAGAAGGGCGAATTCGCGGCCGCTAAATTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGACCGTCGTTTTACAACGACATGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCATCTGGCGTAATAGCTAACAGGCCCGGACCGATCGCCCTTCCCAACAGTTGCGCAGCCTATACGTACGGCGGATTAAGGTTTACACCTATCAGAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCGGGG
AG0171(SEQ?ID?NO：9)
CACTTTGCATAAATACTTTTACGAGCAATTTTAAAAAAAAATTCTAAAAATGTCTATTATTTGTGGACTTGGAATAGCCGTTTATCTGCTTTGATTTTGTCGTTTCTTAAATCAAAGTCCTAATCAGGGTCCTTATATAAGATCAGTCCTCTAGAATCTGAATAGCTTTTTAAGACAAAAAAAAAACAAAACAGATTAGAGTCCGAATC GGACTCGAACATCTCCAATTTAACTTCTATCTTTTTTTTTTCTAAAATAAAATGTAAAATAAAAATATTTTAATTGTATGAAAAATTGCATTCAATAGCTAAAAAAAAATAAAAATTCAATACATAACTAAAATCCCACTTT[CTCTCCTCTTTTCTCTTCCTATCTCTCTCTTCTCTCTCTCT]AAAAATCTAATTTTTCTTTTTTTTTCTGGTTATTCCCTAAAT AAGCC CTAATTGTATTCTATTTTCACTCTAAAAAATAGCTTGATTTTATAAATAGAATAATTCATTTGTTTTTTTAAAAATAAATTATCATTAGAATATAATTTAACTTTATTATAAAATTATTCTCTTTTAGAGCAAAAAAATAAAATAAACCATTAGAAATTGTTTTAGAGAAATCATCAGTCAAAATCTCACNNNTTCCACTATTTAGTTTCATCTCTNNNGCAATCAGACGTAAGAACACAAAAACATATGTTAT
AG0203(SEQ?ID?NO：10)
CAGCCATTCTCTATGGCCTTGGTGACCATGGAGATCAAGTGCCTTAACAGCAACCGGTTGGGCTTCTAAACCCGGTTTAACCTTGTCATCAATGAACCCTTTGTAAACTGGCCCAAACCCTCCTTCTCCCAGCATGTTACTTCTTGAGAAATTATGCGTAATAACTCTCAGCTCAGACAAGGTGAACATACGAAGCTTTTGAGATGTGGAGGAGTTTGAGAGGTCATCCATGACCGACATGGGCGAGCTTGGATCACTTATGTCCGATAACGACAGCCTCTTGATCACCGGACAAGTTCTTATTTTCATT GCGTTGCCCCTCTCCTCTACTTCGTATCTACTCGCGTTCTTTGTCCTGTAACATCCTAAAAACAGAGATGTCAATGATGT[CTTCTTGTTCTT]GGTTACTGCCATTTT[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]CAACTTTTTGGAGAAAATGGAAGATAGATAGTGTTTTACT CTTTTTGATGTATCTTTTGTAGA TTGCGTNNNNNTNNNAATTAAGGGTGTTTTAGTTAACCTACTTGTCTTCAAAGTCCTGTATTTATAGNGGTTGTTGTGTCTTATTCGTAGTGACTACCTTGGAACTTTCTNNNGACATATACTGTN
AG0239(SEQ?ID?NO：11)
AAGCAACCTACGGTGTTCTCTTATTACCGAGTGCTATTAGGCTTGTCTTAATGTCCTAAACAATTACGTTTTTACTTTGAATGTGAACCTGCTCTTGAACGAAATATCTTCATGACTTCATATCATTTGTTTAATTCTACTACTGTCTGGTTAAAAATGGTTGTATGTTGCATATATTGCTATTTTAACCACATGT AAGAAAAGAGAAACGATCCCACGTTTATACTCAAATTCAAGAATCCAACACAAATCCTCTCACAATTCTATTAGATTGGAAGAAAGAAACTCATAAAATAATTATAAATAACAAAAGGACAAGTAAATAGAGTCTATCTGGAACATAAAAACACTAACGGGTCTTGTGGGGTTTACAGA[TTTTCTTCTTTCTCTTCTTGACAGATTTGGGTGTCTTGTTCTTCCTAGCCTCTCTCTTCTCTTCTTTTACTTTCTTCTTGAGCTCTTTCCTTGCTGCTTTCTTGTCTT] CANGA CCCAACTCCTCTTCACTCTCACTTCCACTTTCCTCTTCTTCNNNNTCACTTTCTTCTTCTCCTTCTCCTTTTTCTTCCTCTTTTCCCTCTTCAACATCAACCTTTACTNNCTGCTCATCTACTTTAGGANNNCATCCTTANNN
AG0243(SEQ?ID?NO：12)
TTTAGAATCAACACAAGACATAGNCAANTTGATTTTGGCTACTGGGTATTAATAATCCCCAAAATCACATCCTTCAAACCGAACGAACAATCAACAAGAAAATAAGAGCTACGATTCAGAAAAAGCCTATGATCAAAACGCCTAAAATAACNNNAAAAAAANNNGGAGCAATTTTAAACAAATGGAATTGAATAGTATGAGATGAGATGAGAAACAAAAGAGAAAGCAGTGTGCATAGATCATCAGGGAAGCTAACCTGAAATGATTTGTT GATTGGGGGAT GAGATTGTTGGAAGGGGACAGAGA[GAAGAAGAA]GGTTTGCTTGAAGACTCGAAAATTAAAGCTTGTTAAG[GAAGAAGAA]GAG[GAAGAAGAA]GAGGATATAAATTGACATGGACCTATTAAATGCCCATTTTGTTCTGNTTATTTACTTAAGATTGCCACTA TGACCTTTGACTTTTGGACGGCGNNTGTAGCTAAGCTACTGTTTCTTCATTAATCACGCTTGCCATGATTAGTTTTTTTTTTCCTCCTATAGNNTTCATANNTAGCCCGAAATTACTGACTTTTATGAGATAAAGATCGTATTTTTTTATTTCTTANNGTTTAATACCCT
AG0272(SEQ?ID?NO：13)
CTACTTCCAAAAGAAAACATTAAATTAATGTTTATGTCGAATGTCATTTATACTGAACAAAATAACCTTGAAAATATGTTCTGTACGCTAAAGTTGGGAGAGAGAAGGAGGTTGAAGAGATTTTGTCAAGATTGCGAGGAGAAAATTCTGATGTATCAGATGAAGCAGGAGAGATATTAGTAAGCATATATATGCATGAATAATCATATGATCAATGTATATATTTTTTACTTCATAATATTTTGATGATCATCACGCATATACAGAACATGTTAAACAACAACG AGA TGATCGCGGTTTCCTCAAGTTGTTTCAGCGAAAATACACGTTCTCACTTACTGTAATT[CTTCTTCTTCTTCTTCTTTTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]TAATAACCCNNNTGGTTTACACAGAT TGGAGTTGTTCTTATAG CTTTGCCTCAACTTGGAGGTCTTAGTGNGTATTCTTTTTACACTGAGTCCATTTTCATATCTACAGGTAAAATAATTCTTCTTCTTNNNCAAAATATTGATTTTTATATATTTATTTACCTTAACGATAATTGTTGATAATTACNNNNATCACGTGTATCGAGTGATGTTGGATTCATATCGACATCTATAGTTCNNNNNTTACCGATTTCGAGTGACCTTGTTTAGAGTTC
AG0304(SEQ?ID?NO：14)
ACAAACATAATTGCAATTAAACGGATAGTAAGGGTCACAGATCACACAATACTGCATCGAAGTTTTGTCATCAACACAAGTGCGCATCGTTTCATTCTTTTCTTTCTTCCGGCTTACCTGAGCCCGGCCGTGGCACAATCTTCTTCAACAGACAGCGTTTAAATAAAACTTAACTTGGTAGGGCTGAGGATTCAAGAATCATTTCTTGTAATTCACTGGCACATCGTCGTCATCTTCTTCAAGATCACTAAACGTTACATCTTCATCCTCATCCACAA CATGTTTGGTTG CTACGGTGGAGCTAACAGTTCCTGCATTATCTTCATCCTTCAACCAATCATCAA[CATCATCATCATCATCATCAT]CGACTTGAACGTCAATAACTCTAGGTGAAGATCCAGTGACTGGCTTGTCATGGGGCGCTGGTGCTGGTTTTTCTTCAATCACAGGCTTGTCAACAACTTGTATCTCCTTGCTCTCGATTG GGTGCTTCTCCGTCTCAACCTCNNNTGTATCACTCAAATGTATTGTTTCCANNNAGATGNANTTGACTGNNNCTGGTGAAGACACAGTAAGTGGTTCCTCATTGGCA
AG0323(SEQ?ID?NO：15)
AATCATCCTAAATTTACCTAACCCATGGATTCAAAAGGTAACTAACTACTCGCTAATAGACATGATAAACCCAAAACCAATAGTGATTGAAGGTTAATCATGATTAGTGATCAAGATAATCCAATAAACACAAGACAAAGATGAAAAGAGGCTAAAGATTGAATCTTTCACCAAAATATGTTCATGTCTAGAGAAAACAAGATAGATCCTAAGAATCTAACAATACTAAAAGCATGATAGTAAGCCCTCTAAGCGTGTCCACGTAAGTTAATATATTCAGCTAATCAGAGATTACTAGCTATTTTGCCATGTCATAACAATTTTAAGTC GACCAAT ACAAAAACCGGGCAAGGCGTCTGGGCCATTAGTAATATCCAGTGGCCAATACGAAACCCATCTCATTAATATCAAATCTCCAATGAAAGCCATTATCGTGGCGACTCTTCTTTTT[CATCATCATCATCATCCTCATCATCATCATCATCATCATCAT]CGCTTGGGATCACAACAATTTCCTGTT AGCACAACCC ACTCTCCATCAATCAATCAGGGTCTTTACTACTCTTTTCATGCTTTCGTNTCAACTCCCTTTGTTTATCCTCCTATATAAATCATTGAATATCNNNATTTTGATCCAAG
AG0324(SEQ?ID?NO：16)
TA GTCTCACCAACTCCAAACTTGTTAACATCTGAAGGAGTCTGAATATTCTCCTCCT[CATCATCATCATCATCATCATCATCATCATCAT]CACAATCAACAACTTCAACCTTCTTCTTATTGTTATCATCATCCTTTACTAATTTCTCCTCATCACAATCAATAACTTCAATGACTGAATCTTGAGAAACTGCTTCTTCTTCTTCTTCCTCCAAATCGATAAA CTCTTTATCTTTGGTAAGGA ACCTGAAGGCATTCAAAGCCGATCTCTTGGCGTTATCATACTCGCGAGTGAAAAGCTCCCCCTTCTCGCAGCAATCGTTAGGCTTAGCCGTGGGTGATCCTCCGTATATAAGGTTCGCTCTGCTANNAGCGTGGATTGCTCGTCTAAGTGGAGTTTTGGCGTCGGGATACCTCGAAATCCTCGAAGATGGCTTGTTGGGATCCTGAGACATGGTTCCGAAGGAGAATCTGCGTTTCTTCGGAGCTTGGAAGTAGGGCGAATGTCAGCGGAATTGGGCGGTGGAAGGGTGGTTGGAGTATAAGGAATCTTCGCTGCGCTTGCGATTGATGGCGACNGCGCTCATTTNNNGAGTCGATCACTGAACCCTANNGATTGGGAGATCGACGNNNGGAGAGGAACCATAAGAGTNGAGA
AG0328(SEQ?ID?NO：17)
TTGCCAAAACATTTAACCAGGTGAGCACTTAAACTCTTGTCTGGCCCAAAAAAAAAAGAGTGAGACTGTATAGAGGATCAAGCCAACAGTAGATGAGGAAGAGGAAGGCCATGTAATCTCTAATCCACAACGATCTTGATTACCCATATAGGTCCATTGACTTTGAATCTTAATTTCAGAACATCAACAAATCTTCATCTTTACTAAAATTACAAAAAATCTTTTAACTTTTTAATTTTTGAAAAAAATACATATACACACATACAGCTAGTCTCTTACGAAACACTACACAACTAGATAACTCCAAACATTTACAACTGAAAGTTTATCAGCTTGGAAAATCATCACTCAGATTTCTTGTGGAACTTCACGGAGTCTATCAAGTGTATTAACAATCTCACTCAGACAAGCGATG AGCTCGTCTCCTTGCTGTC TCACAAACTTTAACTTGTCCTCAATGCCAACATCATCCTCTGTTTCACTTCCATTTGCTAGATTTCTCCCGACTATTGCATGTATCATGTCAGCTTTCTCACCTAGCTTTGCTTCCAACGCTTTAACCTCTTCTTCTATGCTCATCGTTT[CTTCCTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]CTACATCANNNGAAATTTCTAC CACCGGCTTCTTCTTCACTTCCATGGTTTTATTCTTCTGATGGNNNGCTTTTGCACG
AG0359(SEQ?ID?NO：18)
CTCTTGCTTAGNTCTGGACTAACCCATATCCGAAAAGNTTCGAGGACCGACTCGAAAACCCAACCCGAAGATCTGCACGCCTAGGCCTAGTTTACAAGCAAGCCTACCAAAATATGTCAACTCGTTAAAAGCCTTTTAACCTGTCTGGTTCGGTGCACGGTTCAATTCCCGGTTTAGTTGTAACCGGTTTGTGAT TGCTCAAAACCCTAGTCGTCACCCTTTTTTATCATTATTGTGAACAAGTAGTCACCTCTACAAGTAAAACCTTAAACCCTATTGAGCGAGTAGCAGAGCGCAGCAAGAAGAAACAAAACCAAAATATGAGACCACCACGTGGCGGCGGAAGCTTCAGAGGAAGAGGAGGAAGAGATGGCAGCGGACGCGGAGGTGGCGGACGTTTTAATCGTGGAGGTGGCCGCTTT[GGTGGTGGTGGTGGTGGT]GGCTGGCGTGACGAAGGACCTCCCGACCAAGTCGTNNNTTCGTTTTCTC TCCTCTCTTGGTTTTCGCTCTCACTTTACAGCTCAAGCAGAAGTCTTTATTAACAAAAGTTGTCACCTTTGACAAATTAGTCTTATCCCTTTGTTAGAGTCATCTTTAAGTTAAAGGTATAAACTTTGTGAAGTTATTCGTTATGACAAAGTTTCTTTCTTTCGTTGGGTTATAACAGAAGTTGCAACGTTTGTTCATGCTNN
AG0369(SEQ?ID?NO：19)
TTTTGAAGGAGGTTGCTTGGGTGATTTTTGATGAGATTCATTACATGAAGGATAGGGAGAGAGGGGTTGTTTGGGAGGA?GAGTATTATTTTCTTGCCGCCTGCTATTAAGATGGTTTTTCTTTCGGCCACGATGTCTAATGCTACTGAGTTTGCGGAGTGGATTTGCTATCTGCATAAGCAGCCGTGTCACGTGGTGTATACGGACTTTAGGCCCACGCCTCTGCAGCATTATGCTTTTCCTATGGGTGG GAGTGGGCTGTACCTTGTAGTTGATGAGAATGAGCAGTTTAGAGAGGCTAATTTCATTAAGATGCATGATACTTTCCCAAAACCAAAATCTGAGGGGAAAAAGAGTGCAAATGGCAAATCA[GGTGGTAGGGGCGGCGCTAAAGGTGGTGGCGGCGGC] GGTGGTGATTCTGATGTTTACAAAATTGTAAA
AG0370(SEQ?ID?NO：20)
TAAAAAATACCTTAAATATATAAAAAATCTATCTTTGTCGAACAAGTAAAAAATTTAAAACATCTTACTTTTGGAAACGAGGGAATATGATATTTTGGAATGAATCAAAATTGACAATCACCTTGTATAGAACCCATAGGTTCGTGGATTCGCGGTCCTCACCACTAATAGGTTGGACCTGGTTAAGAATCCTTGCACACAGAAATAAACTTAACCATTGCCGCCTTACATTGTATTCCAAATTTGTTAATTACCGGCCAACAACACAATTATGTTATC TCCATTATT ACAACCACCCGCCGCAATAATTATCTCAATCA[GTTTTGTTTTGTTTT]TATTTATATTCAAACGGAATATCGTTATTTAATTATTAGAGCTTTAAATAATCTATATAAAGTCCATACAATTTTTGTTTATGGAAATAGACACTACAAACGCGTATTTTCAGTTTTTTTTTCATATGGAGAGAACTACCAATCAGTTGAAAGAAAAAAGAGAACTACCAATCTACCATATAATATAAAAACAATAATAGTATTAA AAAAAGGAGAGGGCAACGGAACGGGACGGAAGAGAATGGAAATGGTTACGTTTATAATAGCAATGATCTGTTGAACAGCTTATGACACCTCACTCTGCGCTTGCTTCCATTCCTCATCTCTCTCTCTCTCTNNNAACTCTCTACGAAACCCTACCTTCTTCN
AG0378(SEQ?ID?NO：21)
TACAAGACAGACGGACATAATAAGAAGAAATGCAAATAGACAAGTCCTGAGAGAGAGTATCCAAAATAGAGATTTTAAAATCTGTCACTAACTTTTAGGCATAGCTTGTTTAGTTCGCTTAGTCCCTCCTTTCTTCACAAAACCAAAACCAAAAAAAAAAAGATGAGAGAGAGCAGTTTGTTGATAAAGAAGCAAATGAAATGTAACTTACTTTTCTACCGGCGGTGGTG GTTGCTG GTGGAGTTGCTGCTGCAATTGAGTAACGTCACAACAAAAAGGAAGATGGAAATGAAAAAAAGGAGGATTCAATGGATATCAACAAAAACGTGTTAGAAAGACTCACCACTCTGTTGAGCTTCACCACCTTGGCTGCCTCCTCTTCTCGTCGGGGTCTACACATTTCAATAGATTCGTCAACAACAGTAACGAGATTGCGTCGAAACCTAACTCAGAAAAAAAAAGAGATGACGTACCGCACTCGGACCTG[CCGCCGCCGCCGCCG] AAGATGAAGCAGTCGCATCTA CAAATTTCAGATGCCAAATTAGGGTTTAACCTAGAAAATAAAAATATCAATAAGGCAAAGAGAGAGAGAGAGAGTACTAGTTGGATTGCGATCT
AG0391(SEQ?ID?NO：22)
GTCTTCTCACGGCCGTACGGATAACGCCGTGAAAAACCACTGGAACT CGACGCTCAAGAGGAAATGCTTAGGCGGCGGTGGAGATGGTAATCTCATTGTGATGAGGAC[GGAGGAGGAGGAGGA]TCAGGATCGGCGGAAGAAGAGGAGATCGGTGAGCTCTGAG TCTGCTACTCCGGTGGACACTGGGTTGTACATGAGCCCGGAGAGTCCCACCGGAATCCCATCTCCGCCGTCTCCGGTTGATGCTCAGCTTTTAAAACCAATGGCGATGCCGTCACCGGTGGAAATGTCTTCGGTGGAGGAGGATCCGACAGCGTCATTGAGCCTGTCACTGTCACTTCCTGGTCCTGATGTCAGACAGGAGTTGAAGAACGCGGGTTCGAAACACAACTCGTTGCTGTTTCCCCGGTTTGGGAGTCAAATGAAAATTAATGTTGAGGAGAGAGGAGAAGCACGTGTTGGACATAAAGCTGAGTTTTTGACGGTGGTGCAAGAGATGATTAAGGTGGAAGTGAGGAGTTATATGGCGGAGATGCAAAAAAATAGCGGTGGTGGCGGTGGTGAATTCATCGTCAGTGGTTTTTATGATGCCGGCAACGGCGGTTTCAGGGATAGTGGG
AG0410(SEQ?ID?NO：23)
AAAAGGAAAGTTTAAGACTTTAAGCTTTACCTGA TGATCCATATC GGGGAAAATCGCGGCGAGGTGATCGAGGAGAAGC[GAGGAGGAGGCAGGAGGAGGAGGA]GGGGGAAAACGCGGCGGAGAAGAGGATGAGAAGTAACGGAGTTTCTTGGAGACGGGAGGGGAAGAAGCGGCGGACAAATCCTCGAACAGAGATCTCTTGCTACCGCAAACAATCGCAGACATGTTATCTGCTTCCACCTTCTTTTTCTTCTTCTTTCTTCCTTCCTTCAGATCTCAACCTTTCCTTTTTGTTTGGTTTTTTTTTTTCCTTTTT]CCTCTAATCCATCTCTGATCTGTT TCTGTCGGAAACCAAGCAAAAAAAAGTCAAAACACATCGGATCTTCTTCCGCATCTAAATAGATCCAACAACCCGGACTCGGATTCAAAT
AG0441(SEQ?ID?NO：24)
AAAAGACGTTGACTTGATTTGATATGCCATAGAGCTAAACCCTAATTGAATTTCAATCAATTAGGGGTAAAGATCTCTCAATCTACGAACAAAAGATCTAATATTTACAGTCAAAATCTACGGAAAA?CACAAAGAAAAGGCTTACGGCGACTGGGCTGCGAGGAGCGCGATTCTGATTACGGATCGGCATCTTCCGATTCTGGATCCGAGCTAGGCGATACGAAAGATGATTTCTT CC GGAAACCTCCGATTGAGTAACAAGAATCTCGACAGAAGTTGTTTCTTCTCAGTTAGAGAGAAGAGATTAGGCTTCGGCCCTTTTTGTGATTTTGAGAAGGAT[GAGAGAGAGAGAGA]GT[GGAGGAGGAAGGAGGAGGAGGAGGAGGAGGA]GCCTTTGTTATTTTGAAAGTTTGAAAATAGATCTTGAGAATAATTGTAACGTTACTCTTGGTCCTCTATATGCTTATTTATTTATTCCACTAATACTTTATAAGGTATATGGGCTTTATATGGACTATAATCT CGGCCC ATCTATGTTAAACTAATCCGTAATTTTCTTGGTTTTTTTTAAACTTGCGCGCTCCTTAATTTGAAT
AG0477(SEQ?ID?NO：25)
ACTTTTTATAACCGACACTTAAATCAAAACTTGAAAAATAGCATCAATTAGATTTGTAACGGAGTATCATCAATCATCAAGAAACAACAATCTTGTAGGTGAGTAAATAAAAGATACCGTGAATAATGTCAACAATCGTAATCTCATACCACTAATACGTAATTAAAGAAAATAATCATATAATTA GGG AGATAATGTTGGGAATCTTAATCGTATAATCAGAAGCGTATTCATTTCATTACAAATTGATTCTCTTGTCATTTGTTATAT[AATAATAAT]AAAAAAAACGTTAAATCAATTCAAACTAAACCTT[CTCTCTCTCTCTCTCTCT]TTCTATTTCGCTCATCATCATTTTATCTGATGAATACGCCCAATTGAAATCCTTTCCTTATCAACTCAAATTGAGTTTTCAAAATTATTCAATTTTCGGATCTCCGTAGATTTGCTCGGCGGA GGAGGAGGAAGGATGGCTCAGTTGGCGGCGGCGGCGGGGAGGAGAATAGGGGATTACGCGGTGGGAAGACAAATCGGGTCGGGTTCGTTTTCGGTGGTGTGGGAAGGGAGGCATCTGGGAGATGGAAACGTGGTTGTAATCAAGGAGATAGCCATGGCGAGGCTTAGTAAGAAGTTGCAAGATAGTCTCATGTCCGAGATTATCATCTTGAGGAA
AG0482(SEQ?ID?NO：26)
ACCCAAACGAATTGCTCTG TCCGTAGAAAGAACAGGCTCGGGAGCTGAGT[GGTGGTGGTGGTGGT]GGAGAAGCGACGGTGGACCATCCGGGAACGAGTGCAGCGAGAGACGGAGATCTTGACTCGGAGGAGCTTCCGTCGAGGAGCCAACCACCGGGAAAAACGACTCCGAATCCATCGACGGCGGAAGAAAACTCGGAAGCTCCGCCACTCCGTCGAATCCACCGGACCGTGCACCACCGAACTGAAGCTGTTCCCGCTG[CGGCGGCGGCGG]CGACGGAGAT ATTTTAGTTTTGGCGGCGGTTCTTCTCGGTTTAGCGTTTGCGGCGGCGTTGCGAACAGCGTCGGCGGGACTCCACGGAGGAAGCTGAGCGAGCTCGTCGATGGAAGTCTGAGCCTTTCTGATCAGCCAGTCAACGGCTTTGCTCGGTCGGTCGAAGCCAAGGCGGTCTTGAACGTCGTAGAACTGAATCGCCGTGTGAGCCGATAGCCTCACGCGCCGGTCACGTGGCCCTTTGGCCGTGCAGACTTTGCTGTGCCGGTCTTTTCTCCCCGTCGACCGCACAATGTGACCTCCTTGCACCTCCACTATCTCGTCTGACGCAGCGCGGTGCCTCATTGAAGAAGGCNGNNNGGGTTGAGGGGTGGAGGAAGTGGTGAGCTTCGTCGTGGTCGTCTGCCATTGGTTGAGCATAC
AG0504(SEQ?ID?NO：27)
ACAACTTTGAAGTGTGAATAGAGTAAAAGATTCAATCTTTCATATCAAAAGACTAACCTAGACTCGAACTCACGGATCTCAGCAAGTTCTTTCCCC ATCAAATCCACCCACTGCACCTTCTTCTTCTTCTCCCTTCTCTCACCATCCTCTGAATCTAAAGTTTCCTTTTTTAGACTACTCTTCAGGATCTCTCCATTACTTTGACCTTCCTCTAAGGCACAATCTTCTTCCTTTCCCTCTTCTTCTGTTCCCTCATTCACCAAACTATCAACGTGATCAACAACTTCCTCTACTTGTGCATCAACATACTGAT[CATCATCATCACCGTCAT]CAACAACCGAAGAGACTTGA[GGAGGAGGAGGAGGAGGAGGAGGAGGA]GTGTCCTCCAATTTCAGAGAACCAGA TGCGTAGATGTGAGGAGAAGGCTTACAGAAGCAGATGAAAGAAGGGCACTGGATCTTACAAAGCAAAACCCTCATCAAAACATATGTTCCAATCATCAACCAATTCAACAAGATCTCTTTTGTCTTTGGCAAAGTTAGAAACTTTGTGTGCCCATTGATATGCCCAGATTGAGAAAAGGAAACACTTTTGATTTCTGAATAAAAAGTAGAAACAGAGCAGCAAAGAAGTTAGTATATATCTCTCGTCTTGGATAGAATCCAAAGACCATAAATAACGAGTTGATCAGATGANNNAGCAGACAAA
AG0510(SEQ?ID?NO：28)
ACTGGTCTCAATGGAGGTGGTGGAGGAGGAGGAGGAAGAATACGACTGGAATCTCTTTTAGACCTTGTAGAGAGTTAAAAGATAGTTTTAAGAAACAAATGTCAATCTCTCAAAATAGGAATACTATACTCTTTACCTGTGAGATAGCTGAGCAAAATCA TCATCTGATTCATCGTCATCATCATGATTGATGCTGACAAGTTGAGCT[GGAGGAGGAGGAGGAGGAGGA]GCTGTTGCACCGCCTCCATTTGAAGGAACAGAGGCGATATCGTCATGACGCTGAAGAACACGCTGCAAGTTATCGTTCAATGCTAATCCCTGGCACAGAAGCTCCTCGTCTCTGAAGAAAAGAAACATCATCAAAAGGGGATGAAACATCAGGTGAT[GAAGCAAGAAGAAGAAGAA]AACTCA CGTGGTGGTGTTGACA AGAGTCATCACACGTTTCTGATAGGT
BG0031(SEQ?ID?NO：29)
C GAGGAAGCAT[AGGAGGAGGAGGAGG]AAGCAGTTTGAGTGTTTGGAGGAGATGCCTGAGG[AGAGAGAG]GAAGGGAGGGAGTCGGACGAAGGCGTGTGTTTTTGCACGTGCAGCGGAGGAGAATCCGGATCCGAAGACGGAGAGGGAGAGTGGGAGTCGGTGGAATGGACGGCTGAGATGGAGGCGGAGGCTGAGGGAATGGGATGGGCCGTTG ATTTGGGGATTTGGGTTA TGTGTTTAGGTGTGGGCTACTTGGTGTCCAAAGCCTCAACTAAAACCTTGAGAGGTGGAGGAAGGAGAAGAAGATCAAAAAGTTTCTTTTAGAGTTCTCTGTAATCAGTCAGTCTAGTTGTTCAATAACGTTCTAATGTAATAGTACAGATCAATAAACCATAAATGTAAAACAATCCATGATTTTGAATACCAAGAGTCGCACGAGTTCCATTTTATTTGAGAGCATAGAACAATAAACTTTCTCCTCTGACCTGATGAACTAAGGCAAGTTCATGCAAGAATCTAATGAATGCAAGCAATCAAGTACGTCAAATCATATTGCATTTACAAATTATACAAATACACAAAGGATCCAAAAAGTGCCTTCTCCCTTTTCTTACTAACAATAATAATAATGCAGCAAAAAGGAATAAAAGTTTATCAAAAACGTGTGATGATAATTCAATGTAAATAAGCAAATATGTGGAGAGCT
BG0106(SEQ?ID?NO：30)
GTAGCCTTGTGTGAGTTGGAACCAGACTTTCCTGTTTCCCTGCCTTGTCTCAAGGTTATGCATTTAGAGAGAGTTATAGCTAACCTTGAGAGGCTTATAACTAGCTGCCCTGTTCTTGAAAAGTTAACCATAATCAGGGATTCTTTTGAAGTTCT CGAAATTATGTGTGTGCGCTCCAAGTCTTTAAAAAGTTTGGCTCTACTGATTGAAGCTTCTGATACTGATCTCTTAGAAGATCACGATTTGGAGATCGATGCCCCAAAGCTTGAGCGTATGAGTCTCTGTGATCACTTATCCAGAAGCATCGTTATACACAGTATTGCTCCCTCTGCAGTGGTACAGATCGATGTTAACTTTAAT[AGGGAGGG]TGGTGATACATTATTGGACCAA[GATGATGATGATGATGATGATGATGAT]TCCAAGAGAACTA TG ATCCGTAATTTCCTAACCGGGATATCCACAGTCAGCCTCATGAAGATCTCCTCTGATACTCTACAGGTAC
BG0111(SEQ?ID?NO：31)
GTAATCATTTCTTTGTTATCTCTCTTTCCATGATCGTCCGTCCAAGAGATATGTAATTGGCGTTGTTTGATTCTGCAATCCGTACAATCCATTTCTAGCTGTTAATCTGAATATAGCCATCTTATTAGACTGAAATCTAAGCGCCTGGATGGGGTGGTTTTATTT TCATTTTGACTTTTGGCGTTTGGTTTTCAGATCTTTAAGATAT[GATGATGATGATGATGATGATGAT]GAAAATGATGAGATTTAGATTTTACTG[ACCACC]CTTTTTTTTTTTTTGTCTTTACGTTTCTTTCAGCTCAATTCAGAGAA GAGCCCTTTTCAACGTACTTATGCAGCTCAGGTAAATTTCATGTTTATCTGACACTTGTCTAGTAATGTGTGATACAATCTAAGAATGTAAATCTTACAATTGTGATAAAAATATTCTCTCTCGTGTTTAGATAAAAAGATGTGGAGAGATGGCAC
BG0119(SEQ?ID?NO：32)
GTGCGGGTTCGAGCAGCTCTCAGCGCTCGCGGAGGGAGGCATGAACGTGGCCAGGCTCAACATGTGCCACGGCACTCGCGACTGGCACCGTGACGTCATCCGCAGCGTCAGGAGGCTCA ATGAGGAGAAAGGATTCGCG GTCGCGATCATGATGGATACCGAAGGTAGCGAGATTCACATGGGAGATCT[CGGCGG]CGAGGCCTCGGCTAAAGCAGAGGTTCCTTCCTCTTCTTGAAATCTT[GATGATGATGATGATGATGATGAT]GCAT[GTTGTT]AATCAGATTATTGGATATAATCCGGTTTAGTTAGAGACCGGTTTAGTTAG[ATTAATTA]TGGTTAAGTTTCTTTTTGCTTAATCATGTATATAAAGAAATGTTAACACAGATGAGGTTTTTGTAGGAT GGTGAGGTTTGGACGTTTACCGTTAGAGCTTTTGATTCGTCTCGTCCTCAACGTACCATTAGTGTGAGTTATGATGGTTTCGCTGAAGGTAATGTGTCTTTTTTTTTTGTGTTATGAAAGCATCAAGTGGATGTGAGTATGAGATGGGGATCGATTTTTTTTTTTTTTTTGTGATTTCAGATGTAAGAGTTGGTGATGAGCTTCTTGTTGATGGTGGAATGGTTAGATTTGATGTGATTGAGAAGATTGGTTCCGATGTGAAGTGTCTGTGTACTGACCCTGGGCTGTTGCTTCCTCGAGCTAACTTGACTTTCTGGAGAGATGGGAGTCTTGTAC
BG0181(SEQ?ID?NO：33)
GTAACATATACAAATACTTCTAGGAATCAATCGAAATATATATTTCATATCGCAATTTCACAATACTGTTGAACTTACAAACGTGTATAATTACACCATTTTTTTACACAAAATCTTTAACATGTCGATTTCTTATACCATTTGTAATTAACTCAACATATTTTTTTAACTAAATCAGCCTCGCCAATTTGTGTTGGTTTACGGAACCGGTACAAATATTGTTGGCCTGGCCGTTATTAATTTCAAATGATTGATTCATAGGTAACATGAGAAGTTTGGAGAGCTTACTAACGAAAGCAGGAGCGGAGACATTGCCATTG GCAGAGCAACGAAGTACGC CTTGAATATTGATGAGACCTAAAATGCCTCCAAGGAC[ACCACCACCACC]AAGAATCCCACCAAGGCCACCATTACCAAGAAGCCCCCCTACTAGGCCACCATTACCAAGAAGGCCACCTAGGCC[ACCACCACC]AAGAAGGCCACCTAGGCC[ACCACCACCACCACCACCACCACCACCACC]AAGAAGGCCACCTAGACCCCCAAGCTGAGCCTTA ACCATTGGAGACACCATCACG AGACACACGAAGATCAGTGAGAAGGTTATGCGTTTGTTCTCAAGCATTGTCATGTTCTTGG
BG0228(SEQ?ID?NO：34)
GTATCTATCTCCTCTTGCCTAAATCACACCATGACTGACTTTCCCAAAATAACCTAGAGATCCAGAAAGAACGGAGGAAAGAAAGAAAAAATGGAG GAGACGAAGCCATTGGTAGGGAACCATCCCCAGCAAC[AGCAGC]AG[CAACAACAACAACAACAACAGCAG]CTCCTGTATCAACACCAATTACAACAGAGACAGCAACAGATGCTTCTATTACAGCAGTTGCAG AAACAG CAACAACAACAAGCCGCCATGTCTAGGTTCCCCTCCAACATCGACGTTCATCTCCGACCTCCAGGGTCAATCCAGACCCGACCAATTGTTCCCCCTCAGCAGCAGAACCCTAATCCCAACCCTAGCTTGGGACAGCCTACACCGAATCTTCAGCAGCAGCAGCAGCAGCAGCAACAGCAGGTTGTAGCGAGTCAGCAGATGCTGCAGCAGCAGCAACAACAGCAGCAGCAGAAGTTGATGCGTCCTTTGAATCACATCGAGCTTCAATTCGCTTATCAGGACGCTTGGCGTGTCTGCCACCCTGATTTCAAGCGACCTTTCTCTTCTCTCGAAGACGCTTGCGAAAGGTTCAGTTCTAATTTTTATCTAATTACATTTGTCTTTTTGAGATATTTCCTTAAATAAAATCGGTTATAGACAATCTCATCCGTTCAATCTTATTTCAGGCTATCGTGTGATATATGCATACGGGTCTTGTGATCTTTGAAATGAAACATTGATCTGTTAATGACTTACTTACTGGTCATATCTGCAACTTGTATGTTCTTCTTTAGTTCGTGTTTGGTATTATGGTGATGATATCTGTTAGCCTTTTCGTTAATTTCTATACTTCTTTTCATTGATATTGTTTGTGTTAGATCCAATAGATCCTGCTTCTTTTGGTGTTCGTGCGAAACTTAAATCTCTTTCTGAGTTTAGTGTGGTTGATTTTATATTATTTTTGTCATCTAATGTGGTTGATTTAGAATTACAAAACTTTGTGATTGTTTCCTATTTTAGTATAACCACCTGATTCACTGATACTGATAATTATTCCCTGACTTTTATATTTATGCTAAAAGTTTACAACTTTACATTAGCATATTATTGGTTTTATTAGATACATTTGTTGCCTTGATTGAACATTTCTGTATATTGTTTGTTTTATCTTACCTCATAC
BG0255(SEQ?ID?NO：35)
CTAGTGGCTACAAATCCAACTGTCGGTTCTCACTTGGGAGACCCAGGTTTGGATCTATCAAGTTTAAAAAT CCAAACTCAGCACAGCCTTTCATTTCTGAAACAAGAAAAGAGATG[GAAGAAGAA]GATCAAGAAGCCAAAAGCATCGGTTTCAGGGAG[GAAGAAGAAGAAGAAGAA]GATTAT[GATGAT]GGAGCTAAGGGTATTGATCTAGAAGGAGAAGAGAAGAAGCATAT A TGCTGTGAATGTGGCAAACGTTTCAAGTCAGGCAAGGCGTTAGGTGGCCATAAAAGGATCCATGTGCTCGAAACTCGCAAATTCTCAATGGTGAGACCGAAGATGGTGGTGACGTCTGGTGCGGTTGCGGTTGCGGTTGGTAGATCTGATGAGCAGAGAGATGATTTCGAAGTTGATTGCTGTGTTTGTCATAAGAAGTTTACATCGATGAAGGCTTTGTCTGGACACATGAGGTTTCATCCAGACAGAGGATGGAAAGGTGTTTTGCCTCCTCATCATCCACTTGATGATCATCATGGTGGGGAGTTTATAAGCTCCGATTACGATGATGATGCTGATTATGATTATCATGAGGATGATGATTATGAGAACTCGGAGTTATGGGATATTAATCGTTGGGAATTGGACAACGTGGTTGACCTTAAGGACTCGATCAAAGAAGGATGGACGGTGACAGGAAAGAGAGGAAGGAGAAGTGCTTTGAAGATTGATGAACCTGATGATATTGATGCTAAGGATCTATTGTTCTTAGCTACTACAGCAGAATCTGTCGATGCTGCAGAGACTTGTTGTGATTCGCTTTTGGGGGAAGAGATGATGATGAAGAAGAGGAAAAAGAAGAAGAAAAGATTGTCTGAGATGGAGAAAGAGTCATCATCTAGTCATGGTCATCATCAGCTTGAGGTTGGTGATGCTGCTGAGGGAGGTGGCGGTGCAC
BG0278(SEQ?ID?NO：36)
GTATATGTCTTTGGTTATTTTTTTTGGTATCCAAATAACCGTAAATAAAAATTAAAAATGGCCCGTTTTCCCTCGGATAAAAAAATTGTAGAGTTTAAATCATGTCTTTTAAAACCATGGGAGCAAAATCAAAGGAAGAGAGAAGATAAATTAAATGG TGGCTGTTCAGTTGTTTAGCTGGAAGACATTGATTCTTCTACCTTCACAAGCTTCAAGACATAAGGGTTTCACTTCTTTTAACAGGTTTTTAATCTGT[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTATTATTATTATTATT]ATAATAGTTTCAAGTTTCTGAAAAACAATTGATTCCATGGTGGTGCATGTGTTTTACAAGATATCTCACTGAAAATTAACTTTGTTGCAGAACATTGAGTTT GCACTCTCTGCCTTCAAATGGGATTGATTCTTTTAGATCCCGAGGTGAGGAGGCTCTGAAACACATTCCACGTCTTAATGTCCTTCCTCTCAACAAAGACTCATACTTTCATACTATCATATTTTCATAATTTCATTATTACAGGAACCTTCAGAGTCAAATCTCAAAAGACAGGAGACACAGAGTCATCTACTTCCAACTTGAATCAACCTAATGATTTAAAATCCAAATTCCATAAGGTGCGTGTGTGTCATGCATGTCTTTACTTTTTTTATCTAATGATTTACTTAATGCTTTATGTTATAATCTTTCTTAATATACATATCTGCAGAGTCTCCAATATAAACTTGTACTAGGATGCATCCCACTGTATGCGGTATCGAGAATTGTACAAAAGATCATTCATGGGCTTCCACTCCACATTCAGAACTCAGTAGGGGCTGGCTTGCCTTTTGCTTGTGCATCAGACTCTCTGAATAAACCATCTTTAAGTGGTATCAAATGGAGTCTTGCAAGGTTCTTTTTCCTGTTCAATATTCGGCTCGAGAAGAACGTTGCTAC
BG0295(SEQ?ID?NO：37)
CTGTTCCGTTTAACTATGCTCGCACCTCCATTATCTCTCCTCTTTCATAACTCTCTCTCCTCCTTCTTTCTTCTC CACATCTCTCCGATTTCATCGCTAGAATTCTCCACCGATTCTTAAGGTATGTTTTATCTTCACTTCAACTCTTGTCGGAATTCACTCTCCTTGCCTGTCTGAAACTTTCCATTTGCAGATCTGTAAAACTTTCTATTTGTGTTTCCTCCTTTCCGTAGATCGAGAAGAAACGATGACTTCAACGG[AGGGAGGG]ATACGATCCCTCTTGTCTC[TCCTCCTCCTCCTCCTCC]TTCTCTTATCCATAACCACTCTAATCTCAGCCGCTGACTACA CACCCACCGACAAAATCCTCTTAAACTGCGGCGGCTCCTCCGACCTAACCGACACAGATAACAGAACATGGATCCCCGATGTCAAATCCAAGTTCCTGTCTTCCTCCGGAGACTCCAAAACATCCCCCGCCGCAACACAAGACCCCTCCGTCCCCACCGTCCCTTACATGTCCGCCAGAATCTTCAGATCTCCCTTCACTTACTCCTTCCCGGTCGCCTCAGGTATTGGTTCAATCCTGGTTTAGTAATTGTACTTTGGTTTACTCATTTCCGGTTTACTAAACACTTTTCCCTATCACAGGTCGCAAGTTCGTGCGTCTCTACTTCTACCCCAACTCCTACGACAGCCTCAACGCAACCAACTCCCTCTTCTCCCTCTCCTCAGGACCCTACACTCTTCTCAAAAACTTCAGCGCCGCTCAAACCTCCCAGGCGTTGAACTACGCTCACATCATCAAAGAGTTCGTAGTCAACGTCGAAGGTGGGACCTTAAACATAACCTTCACACCAGAGTCAACGCCTTCTAACGCCTACGCCTTCGTCAACGGTATCGAAGTAACTTCGATGCCTGATATCTACAGTAGCGCCGACGGGACGTTGACCGTTGTAGGGACTTCTAGTGGCGTCACGATCGATAACACCACCGCTCTCGAGAATGTCTACAGGCTCAACGTCGGCGGGAACGACATCTCTCCTTCTGCTGACACCGGTTTGTTTAGGTCTTGGTACGATGATCAGGATTACATCTTCGCCGCGAGTCTCGGTATCCCCGAGACA
BG0452(SEQ?ID?NO：38)
GGTCTGAGATATATCCTCGAGGGTTGTCCTAAACTAGAGAAGCTTGGGATCAGGGACAG TCCCTTTGGTGATGTTGGACTGCGCTCTGGGATGCATAGGTATAACGACATGAGGTTTGTTTGGATGTCGTCATGTCGGTTATCCCGGGGAGCCTGCAGGGACATTGCTCATACTCTGCCTAGTG[TGGTGGTGG]AGGCGTTTGGGTCA[GATGATGATGATGATGATGAT]GA CGAAGA CGACAATGCAGATTATGTGGAGACGTTGTACATGTATCGGTCCCTTGATGGCCCAAGGAAGGATGCTCCAAAGTTTGTAACAATTTTATGAAGACAAGCTTAGAGAAAGCAGGAGCTGAAGTAGAAGAGAATGTGTGTTTGTATGATTGTTTGTACCATTTGATTTGATTGGCTCCCCTCTGTTTTTGGATTTGTCTTGTACCAAGAAAGAGTGAAGAGTCAGTGAAGAAAGAGGTTGTTTGTGGAAGTCAAAGAATGAAACTTTTATTATTTGTGTGTAATCAAGAATATGATTTTACAGCCATTTCACGATTATTTTTGTCTACAAGAAGTATTGGTTATACATTACATTATAAGATCTTCACCAATCTTGACTTCGTCCTCCATCAGCAGATGCTCTAAGGTGTCGATGAAAGCAGTAACTTTCTCCAAGCTCTTCTCATCAAGCCTTGGGACCGTGTGGCCCTTGGGATGATGGACCACCACCGGATTCTTGAAGGAATCTATCAGCTCAGTTCCGTAAGGTTTCAAAAAATCAGTCTCTCCTGCAAAGAAAAAACTCATTTTTCACATTGAAATTTGCAAACCAGATATACAATTTAGTAGGTCATCAAATTACCTAGAAAGTGGAGGGAGGGAATGTCCATGGTAGACGAATACGCATCCTTCGCCACCTTGGTGGATTTGAACATAGCTCCTCCAATAATTATGATAAACTTGATCTTTGGTACTTTCTGGAGTGCAATTCCCTGCAATATAAAATATAATTCTAAGATAATGTAATGCGATTTCCCAACGCAAAAGCAACACTACTGACGTACCTTAGCTTGCAGTCCTGGTAATCCTCCAGACAATATTGCACCCTGCAAAATTAACATAGAGATATATTATTAGATCTTATATAAGAAACTGTTAAATGAGAAATGAAGCAATTTTGTAATTAGAGTACCTGAGAAAAGCCAATGAGACCATCAAAGGGACCAAGCTCGATCATACGATCCTCTAAATACTCCAAACATTTCTCGAAATTCG
BG0516(SEQ?ID?NO：39)
GATGTGTTCTTCATTGTATCTAGCAGAAGCT TGGTCAACAGAAAA TGGCCTGAAACAT[GATGATGATGATGATGATGATGATGATGAT]GAGACTATAAAACTTAGGACAAAGGTA[TAATAA]TC[TTGGTTTGGT]TTCTCTTAGCTCACCTAGATGGTTAGTTGCGAATTGCAGCTCAATATTGTCCTTA GAGAGCATGAAAGGACATGCCATTACCCCAGCGTTGTTGCTAACAAGATTTGAGAGATTACAAAACATTAAAACCGTCACAAAACACTAGACATGAACTACTGTGTTTCGAGAGCTTACATCAAGATGTTTAGTGGAAGACCAGTAGATTTGTAGTCAGATGCAAATCTCCTGACAGATTCAATTGAGCTGAGATCTAACTCCATGACGTCGAGTTTAGCACCAGGGACTTGATTGAGGATATCTTGCTTAACTTTAGCACCGGAGACAGTGTTCCTCACCGCCATAACC
BG0647(SEQ?ID?NO：40)
GCACATATGTCCGCACCTGTACAAAACCGCCTCGACCTGCGTTTCGTCGCAGACGCAGCATTTGCGTTTCATTGGGTTTTCTCTGTAAACCGATTGTTGCAAGCTCGCGTTAGCATCCAAACACGTTTTGACAGAATCTCGTAGTAAGGACATTTCTTGTTGAAGCTGTTGGATCTGTGTTCTCATATCGGTTATCAGCTCCATTTCCTGAAAACGTTTTAAAGCGGTTCAAAGATTTTACTATTCTACTAGTTGGGGTTTGCGAGTTTTCTATGCAATAACAAGAAATCGAAAATTACTTACATGTGAAGGAGGATTGTGAACAGACAAGACAGGAGTGGAAGTTACTTCGGTGTCTTGACAACTCCATGATCCTGCAGGAGACG ATGCAAAGATGGGCGAAGAAGACGATCTGCTTGAGTCATCTCTATCGTTTTGTTCTTCACCTTCCGTTGATGGCTCTTCTTCAGTTTCCTCCGCAGTGTCATCTCTATGTTCTTCCT[CTTCTTCTTCTTCTTCTTCTTCTT]GTTGCAATTCCCATGATTCAGAATGCTTTTTCGAATGTGTCTGCAGACGAGACATCATGAGCCTATCGAT CTGATCTCGTAACCCGCTCTCGAGAAAGTCTGTCACTGTTCTTCTGTAATAGCAAGAAAATATTTATCTTCTTAGTTAATGGTTTAACAAATAAGAAAAGGGATTTGTTGAATCGATGTTGCGTACCGCTCAAGGAGTCT
BG0651(SEQ?ID?NO：41)
AA TGCATAACAAAAGATTTGAACCCGGGTCTTTGGTCAAACAATAATCATCCTAAATTTATGCTAATAGTGATTCTTTTGTTAGCCACTGAACACAAACTCT[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]CCTCTTTCCTCTGCACTCTCTCCGACACAAGACGGCGGTCAACGGAGTCCTTGTCGGTCAAATGATCCCTAAGGACGAAGGAGGAGTTGTGGAGATTTCCGA TTC TGTTCCGCTCTTTTGCTCCAACCTCGCTCTCCTTCCTCCTCTCTAGATCTCGCTCATCATGGTCGCTCTCACTACATAAGTTTTTGAAATTGAATATTGAAAAACTTAGGATCTGAGTGCACTGTTGCGAATTCTCAATATTGTTGTTCTGTAGCTGTGTTTGGGAGAGAGGCAGTGTCTGTAATAC
BG0713(SEQ?ID?NO：42)
ACGGTTCAGCACAGTAAAAAAAAAGTTTTTTTGACTTTTTTTTCTTTG ACCGCCAAAGAAGACGAAAATGAGTCTTTGAGAAAATCACAAAAAAGAAGAAGAAGAAAAATGAATCCTTTTTGTTTCTTCTGCACAGAATCTTCTT[CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT]TTCTTGAGGTTTCTTTTCCTCCACGATTCCTCGTCCCTCTTGCTTCTGTGTGATCGATTTTGGTGAAATTGAG CTGAGTGTATCTGTCCGCCGAGGCCTTTTGTTCACTGTTCAATTCAACATCAGATCAATTTTAGGGGCTTTCAGTCAAAGATCGCTGCTTTGGTGTAAGTTTGAATTTGGGTAACTGAATGAATGTGATCTTTGGTTCCAGTTCATGTAATTATGTTTGATTGACTGGGAAAGTATCATCCTTTATTACGGATTGTAAACATTTAAGGTTGAATCTTAACATTAGCACCATTTGGATTCGAATTTGTTTGGTGGGTTTGGCTTTAGATCCATAAGCAAGCTTATGAGCTCTTAAAGTTATGTTGTTTTTTTTTGCTTAAGCCATTCAAACTGATGAGATATACTCTCTTTGTCTTGCTTCCTAGGTTTGTGATTTTAGTATAGAATCCTGTTATCATGGATGAACACAATAGGAATCCATTTGCAAGTGCAAGCGGAAGAGCAAGTGGAAGTACAAGTGTGAGTTCCAACTCCAGTTTTAGTAGCAGCGTGGCGGATACAGAGGATGATCAAACCATTGC
BG0864(SEQ?ID?NO：43)
TGCAAAGGAAGCAGGTGTAGCAGCTCAAGCTTATGAAGCTCTAAAGACACTGAGAGAAAAAAAAACATCTGCAAAGTGGTAAACAAACTCTTCTTATTTCACACAACACATGGTAAAGAAAATACTTTTTCATGGAGAATAAGAAGAAGAA GAAGCTAAATGCGTTGCGTTGCAGGTG[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]AGCGCATAAGTAGTGTTTGTGTGTTGGTGATTTTCTATTTGGAAACTCTTTTGTAAGCAATAATCTCAGATGCTAAAGCCATTGTATTTAT TG CTCACTTCATTTTACAGCCAAACTAAGTTTTAAAAACTGAAAATATAAAACGCTAAAATTTTCTTTGGTTGACATCAGCATAATATAAATTTAGCTTACTCCCTCGATTCAACAATACAAAAAAAACGACATAAGTTTGAGTTTACATGCTTTCAACCAATAAAATGGAACTCTTTATCATAAAATAACAGTCAACGTATTATTAAGTCCAAACCACCACAAACCAATATTTGCACAAATAAAAGTTTCCAACCTTAGCTGCCACTATAAAGTTATAAACCACCATCCAAAGTCCATTATTTTAAGATAGATTTCGTACGGTAC
BG0869(SEQ?ID?NO：44)
TCGTATAAAATAAAATTCTGAACAAAAATAATTATATAATTTCAAATTGCGGCTCAAAATCTATTATTTTTAAAAACTCAACAAAATTGTATATGGGCCGATGAAGCCCAAGTATTTTAATTACCGTAAAGGAGGGTTT GAG TCGGCCACAAATCAAGGAATTATTTC[CTCTCTCTCTCTCT]CCTGTGACGAGTTG[CTCTCTCTCTCTCTCTCT]CGTCTCGTCCGCGCTCCGAAGAAATTTCACAGATTCCTGTCATGTCTTCCGGCGGAAACTCTACCCTCTCCAACGTCGAAAAGATGTTCTTCTGTTACCAGTGCAATCGCACAGTCACCA TC TCAATCTCCTCCTCCTCCGACGATCCTTTCTGCCCTCGCTGCTCCGGTGGGTTTCTAGAAGAATACGACGAGCCAAACCCTAATCCGCCCCCAAATCTCAACCCTCTCGGTTTCCTCCCCATGGCCGATCCTTTCTCCACCCTGCTCCCGCTCCTATTCGGCTCCTCCTCCTCTCCTCCTTCCTCCACGAACCAGAGCTTCTTCGGCCAGAATCAGCACCCTCCTCGCGGCGGAGCTTTCGATCCGGTGTCGTTTCTCCAGAACCATCTCCAGCACCTGCAATCCAGCGGCACTCACGTCCAGTTCGTGGTGGAGGATCATCCCTCGGATCCGTTTGGCCGGATGCCGGGGAACATGGGGGACTACTTCTTCGGCCCTGGCCTCGAGCA
BG0988(SEQ?ID?NO：45)
AACGGTTTTGTATAAATAGTATATTCTATATATGTATGCATATAATCTTTTTTCGTAACTTAAAAGGATTAAACCGGATTTATTAAAGACACAAATCTAACTTCCAGATGAGAGGTGCAATACACATATGGATTATTTTCCAGATATTTAAATGGACCATAAATATAGACCCATAACCGCGTGGCCACATATGGAACTAATGATTTCGCACTAGAAGGGAATCGATTCCTGACCTGAACCAACAGGACAATTCCTCCTCTAGCGGAAACCATTAAGCCACCACAACATGGTTTTAAACAAAAAATTGTACGCATCTGCGTGGCTTACTATTAAAACATCTCTATCTCTCTCTTAAAATACATCAAGAGTATAATGAGAGATATCTCAGTTTCATGTAGTAAGACAAAA CCCAAGACTCCAACCGGAAAATTCCAACCCTAAGAGGCAAACTAAATTTCATTGTACAATAAAATAATTAATGCTATTCAGTTTTCTAAAAGCAGATTTAAGTCTCTAACTCCAATTTTCCAT[CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT]AAATCCCCACTAGGATTATGGGAA CTCACGTCCTCGTTTAATGCGATTCATGACTCCTCAAAGCCCAGCATTCCCACTCTGCAAATTACTAGTACCTCTTAGTCTTAATTACCATTTGACCAATCT
BG1062(SEQ?ID?NO：46)
GTAATGCGGCAGGACAGCCCTCCTCGGAGTCCACTTCATGGAGGAGCATACTATTCATCCAGTGATGATGATAACCACTCCACCTACCTCTTCCCAGAAATTGGCACC CCAACTCGTTCCATCCCAGTCTCCGCCAACACCACTGTATGAAT[CTCTCTCTCTCTCTCTCTATCTCTCT]TTCACCATTGTTTTTATGATCTTATGGACCTTAATAAATAAACATATGCAGCCTGTTCACCACAACTACCAAATCATTGCGGTGGAAACCTACGAGCA AGAGAAGCAGTA CGAGCCACCGGAGCTAGCGGACGAGTCACAGAGCTTCTCGATCCAGGAGATCGCCAAAATGCGAGGACTCAAGGAAGAGA?GCCAATCGATGATCTCCGAGTCCTAC
BG1090(SEQ?ID?NO：47)
GTGAAACCGGTTCTGGAGAACTCTAAGGTTGTTTTGAAAGATCGCAAAAGAGAGTGGAAGCGGAATTAGGGTTCTGGTTATGGCAAGGAGATGAACCGGAAGGAGGTACGAATCCTTTGGGAAGCATAACAGAAAACGTATCCGGTCGGACCGGTCGGTTAAAACCGTTACTGTTTTTCTTGGTCATCATCTTGAAGTAGTTGGCACGGTGACGAAGACCACGGCGACGAATGCTATGGCGGTAGTAGTAGTAGTGATGGTTGTAACGAAGACGACGGTGCTGATCACGTTGCCGCGGGTTCAAGATAAACGGCGTCATTTTCTTGATGTAGACGAGTTGGTGCGGTGATTCGTCG CCGTTGAGGTAGGTTTCTGCCTTTTGTGTAGATACTCTTGTTTCTTGATTCGATAATGATGAGGATGATGATGATGGCGATGATGGTAAT[GATGATGATGAT]GGTGAGATAGGGAAGACGAGAATGAGAATGAGAGAGATGATGAAGCATTTGACAAGGTTGTGTTTCATCAAAACATCCATTGCGATT[GAGAGAGAGA]GGGAGTAGGACTTTTGGTTTAATAGAGAGAGGGAGAGTAAAGATGAAA CAAAAAGATGTGAGCG AGGCAACTATAACAAATCTTGGTATGGCGTCTAAATAATTCGTTTAGTTATTCGAATTTTAATTAATTTTAGTATGATTTTTGATTGCGTATAATTTGGAAATTAGTTGGGCTTTTTGTTGGTCTGAGGC
BG1101(SEQ?ID?NO：48)
C ACATGCTTGTGGATAAAT[CATCATCATCATCATCATCATCAACATCATCATCATCATCATCAT]CAATATCAAATATATGGTAAGTCCATTTTCATTTAGCTTTCAGTAAAACTGTTAATCTATGCATTCGATAATTAAGAGAATCAAACGAATTGTGTTTGCAACATTATAATTAATGGTTGAAATTCATTAAGAATA TTTAGTTTGGGTTTTCTCATTTTCATACAAACATTATCCATGCATACGGTTGGTCATTAGGTTTTGAAAATATATGAAATCAGAAACATTTTAATTTTTTTTAATGTAATTTGAAAGCATACAAGTTATGTATATTAACTTTGTGTAATTTGAAAGCATACAACTTATGTATATTAACTTTTCAAAATTTGGACTATAAATAAATATTTCTTTGATCTGCCCAAAATCACAAAAGATTCTTTTACAAGATAAACTGTATCTTTTACTCTCTTTTTTGTCAATACTGTATGTTTCACTTGTCACGAATTTGCATTCAAATAACTATGTAGCAGCACATTATGATAAAGTTGGAAGTGTATGAATAAATTGATAATGTAGATTGTAGGGTGAGAAGTTAAAAAAAATGAGTAATTTTTAGGGGCCAAATGTATTTTCGTATAAATTAAGGGTGGAAACATGAAAATTAGATTTTTTATGTCCGAACTACCCACTGACTTGTCCGAAGTCCGT
BG1123(SEQ?ID?NO：49)
GTTCAAAGGCATATTTATATTATATTTAAATTGGGACCAAGATTTGCTTTGGGACAAGCTGTGCCCCACGACTTTCTCGCTAGTGCTCTCTGGTCGCTTCTCCTTCTAGAGACCAACCATTTCCACCAACTCCGTTTTCAGTTCACACCATGCCCACCACTGCATCAGTTAGTTGATATGAGCCCAACTTCTTTCTTCACTGTTTAACAAAATGGACTGGTCAACACAGTCTCTGTCACACCCGAGAATTCTAATGTGGTGGACACAATCTTCACT AGGCCACCTTTTGTC ACCAGTCTCTCTCTCTCTTTTCCTGTTTTGATCCTTCCATAAGATTAAACCTTTATGGTTACTACCATATTATAACGATCTCGGTGGTGGTAGCGTAGCCCAAAGATGATGATCCGAAACTGAATGTAAACTATGTACCAAA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]GG[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]GTAATAATTAAAAC AAATGGGACAAATT AACCCCCC
BG1127(SEQ?ID?NO：50)
CCGCCATAACAAAAATCTTCCCACAAGCGTGTAGAGATCTG[GAGAGAGAGAGAGAGAGAGAGAGA]GCTTTCAAGTGATTAAATCCAAAAGTAATAAAGAGAAGACGGAGAAACTAAAGTGACGCGCCCCCTTCCTAACGGATTATATTTATTTTATTCTTATATATTTATGGGCTTATTGCAGCAATAGCCATATTTGAAATGAAAATTAAGAGAGTAGCCATGATGTTGACATAATGTACTCACTGTCTTTTTACAATTTTACTA GCCGGTTATACCTTTGTAGG AAACAGGTTCCCAGTTCCTTTAACTAAAGTAAACGATGTGGTGATTTACTGACCCATAGTAACAATGAGAGTATTTTAGCAACGCCTAAAATTAAAATGAAAGGAAGGAAAACATTCTATAGAGATGAAAATATAAAAAAAACAGAAGTGTAAAAGAAAGAACGTTACAAACGGAGAATGCATGGATCGTAATGCTGATGCCAAAATATGGAAATAGTTCCACTTCAAATAGAATACACATAGTATAACAATAGTTTAAAGTTTGTCACCGCTATGTCATATGAGAATATTTTCCATTCTATCGGATATAGATCAGTTTATATTTACTAATATAATCAC
BG1149(SEQ?ID?NO：51)
GTAGAGTGATAAGAGAACATCTTGTCTGACTCATGCTTCTTTTCTGCTATGTTAGGCTATTGCTCTTAATGATCTGCCTTCCACTAACGTATCGAGACTGGGGCTTGCTCTTAACTTATCTCTTTTCTACTATGAGACTCTCATATCAACTAAAGCTGCGCGTAAGATCGCAAAGGCGGTATGTTGTTGCTTTCTCTCATTTAGTATTTTGGTTTATGTTATGCGATTATCATCTATTCTCCCAGATGCTCTGTTTGATAAAACTTAATGCTTTTCTTTCTTTTTTTTTGTTCTGACAATCGTTACTGTTCAATTGTATTCATATTGTGGCATAAATATGTATATG TTGCTACACTTCCCTGTGGGTGTGCAATCTTCATATGATATAGTAATGGTTTGCAGATTGCTTATCATTTGGAAGATAGATATTGTAATTGATTATGATGATGATGTGCATAATTTGGAAAGTAGAGCCTATCGTTATTCCCTTACACTAATGGAATTATATATTGATGATGTTCCAAATTTTTTAAATATGATGAATT[GATGATGATGATGATGATGAT]GGTAGGCTTTCGAAGCGTCAATAACA GAAATGCACGCAGTGAGAGAGGAATCATACGAGCAAACTGCATTGATCACGAATCTTATCCTTGACCGTATCACCCCTCTGG
BG1182(SEQ?ID?NO：52)
GTGTTTCTAAGCACTTTTTTTTTATAATCAAATCACATACAGCAAACATAAACATGAGTCTCAGCTTCAAGAGCCAATGAAATTAGCTTCCTTTATAATATTCAAGAACTAACCAGTTTCACTTCTACTAATCCT CGGCGCACT GATGATGTTTCTAACTAAATTGGATACTAAAGAACGTAGCTTTTCACCTCGAATCAAACAACTTGAAAACCAAAACAATCTAAACGAAATTTCATAACCTAAGGAGGATCGGAAACTAAAATTTCTACATCGGAATCGAATCGACGCGAAGTGAAACGAAGATCGAT[AGAGAGAGAGAGAGAGAGAG]GACTCACTCGCCAGGAGAAGACATGTTCGTCGATTTCGAAGATCCGATTGATTCAGAAGCGGAGAACAATCCAAGTTTTTTATTGAGAG AGCACCGAACA AACTCTCTCCCTAGAACGTTCCTTCCCAGCTTCTCTACAAATCACTTGTTCCGCGACTGCGTATCTTATCCAATCATGTCTTGCCACG
BG1197(SEQ?ID?NO：53)
BG1197(SEQ?ID?NO：224)
GAACATCCTGCTGTTTCAGTTCTATTTTTCTTGTGTAGTCAAAAAGACATTATTACCCCATTGGAAATTACAACAACACATTAG TTGTGAGCGCC AAGATAAGGCTAAAGACGTAAAAACGCTCTGAGTATTCATTCTTTCAGGTCAGGTTCAGAAACTAGTTTCGTTT[CATTTCATTTCATTTCATTTCATTT]CATCCACCTCCTCTTCACTTGAGAAGTTCTGTCTTTTGCGATCCTTGTCATTTTTGTAAAGGTGAGTCGATCTATATATGGTCACTAGTATTCTGGAAATGATGCTATTTTAATACTCAGTTCGAACATTCTGTTATCAAATCCGGTTCTAGTT AGTTGTTCGCGGGATAGGGTTTGCTTGAGATCATTTCGCTTCTTTATTTTTTTTAATGTCACTGATGGATCTGGTAATCTTCCTTATCGAATTAGGAAAATGAATCTGTATTAAGTGGACTAATCTCAAATCTAGGTAAAAAAAATGGGAGGAGGAGGAGGAGGAGAAGGAGTTGGCGATTTCAGAGCCAAAGTATGGAGCATGTCTGGTGGGCCTTACTGTAGGCCCAAGCACTGGCGTCGCAACACCGCCTTTGCAATGCTCGGCGTTTTCCTTGTCTGCATCCCCATTGCCATGAAGTCTGCCGAGCTCGAGG
BG1230(SEQ?ID?NO：54)
AGAGAGACCTCCAGTCACCTCGTCTCTTCA GGCCTCTTGTGTTTCC TCCAACTTCCTTTACCAAAAAAAAACAAATCAAAATCAGATTCAAAGGAGAGAAAGAGAGAGGGAGAGAGCACTACAAGAGTGGAAAAGAAGAGAATCAGGTCGTG[GAGAGAGAGAGAGAGATGG]CG[GATGGTGGTGG]TGATGAATCTGAGATGCGATGGTGGTCGTGATGAATCTGAGATGC[GATGGTGGTGG]TGAGGAATGATGGCGGATGGGAAAGATGGC[GATGGTGGTGGTGG]TGACGAGTGAATGAGCGG[TGGTGGTGG]TGACGAGTGGG AG GAGAGATGGCGGTAGTGGTGGTGGTGGTGATGAGGAGGTCAAACCTGATGGATTGGAGGAGAAAAGGAGGCGTCACAAAGAGAGAGAGAGAGATTTGTGTGTTAGGTTAAAGATTGCACATTCAGAAATGTGCTTAGACAATGATCTGAAGTGGTCTTGGTCGAGGTAGTCCGTACATGTCCGTACACAGTGC
BG1241(SEQ?ID?NO：55)
GGATGGGATGAGTCAGCTGCTGGTGATAGGCCCAGTCGAGTTTCAGTTTGGGACATTGAACCAGTTTTAACTCCTTTCTACATATGTCCTCCTCCATTTTTTCGACCTCGGTTTGCTGGACAACCAGGAATGCCAGGTAAAGTCTTTGTACAGTTTCATTTTGCACATCATCTTTGAATCTCCTTAGAGATGGCAATTCTGGTGGTCTTGCAGATGATGGGACTGACATGGAGTCTGCGTTGAAGAGAGCAATGCCGTGGCTTGACAATGGCCTAGAGATGAAGGACCCTTCCAGTACGATATTTCCTGGTCTGAGTTTAGTTCAGTGGATGAGTATGCAACAGCAGAACGGCCAGGTCCCTTCTGCCGCTGCACAGCCTGGTTTCTTCCCGTCAATGCTCCCTCCAACCGCGGCTCTGCACAACAATCTTGGCGGGGCTGATGATTCCTCAAAGTTACTGAGCTTTCAGGCGCCTCCAGGGGGGGTTTCCTCATCAAACCTCCAAT TTAACAAACCGAATCCGCAAGCGGCAATGTCCCAGTTACCTCAGCCACCAACTACGTTGTCCCAACAACAGCAGCTGCAGCAGTTGTTGCACTCCTCTTTGAACCATCAGCAGCAGCAGCAATCACAGCCTCAGCAACCACAGTCGTTGCAGCAACAACAACAACCGCAATCCCTGC[AACAACAAC]AATCACTG[CAGCAGCAACAAC]AATCACTACTG[CAGCAGCAGCAGCAACAAC]AATCTCTGC AGCAGCAGCAGCA ACAACAATCTCTGCAGCAA
BG1244(SEQ?ID?NO：56)
ATGATGATGAAATAGCTCTGAAGAAGAAGTTAATTAAGGAATTGTTGCTGTCTAATTAGGTGTTCTTGTTGTTTGGTTAATTATGTTTGGTTCTCGGATTTGAAAGCTCTGTTAAAGAGCTTCAGTTTTAACTTTAATTATCGGATTTGAAAGCTCTGTGAAGAGCTTTATTTTTCACTTTATCTGTAATTGTTCTCCTGTTCTTGATGATATAAAATATTTAAGTTGTTCTTGTGTTGTTCAGTTATATTTACAGTTGTTGTTTATGATATATCATGTTTCTTTGTCTTGTAGAGAAGTCACGGAGTCCACAGAGATGCTTGGACTGAAGGGAGTCACGGAGTCCACAGAGAGATGTCATGTACCATGTCTTGTAGTGTGCAGGGTCTGTAACGAGTCACGGACCATGTGTTTGTATGTGTCAGTATGTGTTTGTACGTGTCTTGTATGTGTCACAGAGTCCATGTTTTTGTTTGTGTCTGTATGTGTTTGTA?TGTGTCGATGTCTTGTAGTCACGGACAGTATTTTTGTAGTCACGGACTTTTACCAAACTCATCTTCTATTTATAACATCAATCTCATCTTCTATTTATATCAACCTTCTCTCTCGAAACATATA CAACGAACTCTTCTTCTCT GCTTTACAACAACAAACT[CTTCTT]CT[CTTCTT]AACAACAACAAACT[CTTCTT]ACCATATTTATATTTTTTCCCCTTATTATAAACACCAAAACCATCTTTATAAAAACTTTATATGG[CTTCTTCTT]CTC[ATGATGATGATG]CGTTTG[ATGATG]CATTTG[ATGATG]TTTTTG[ATGATG]TCTATGATCAATATTTTGATCAAGCATT TGAGAATTTGACCATTTGTCGTGATCAAGAAGAACGAAGAAAGAAAAGAAAAAAACGAGCGTATATCGAAAGACATCGTGAGGAA
BG1286(SEQ?ID?NO：57)
A GTCCGAGACAGAGTATGCTA[AGCAGCAGCAGCAGCAGCAGC]TGAATACTGCATATGATGCGTCACAGACAAATGCTCAGAATCAGATGCAGAATCTTGCTTCTTTATCAAATGTGATGGTAAGCTACATGTGCATTATTCA TATTTGAAGTGATCCACCAATGACATTCTCCAATGGCATTGCTAACATTGGTACTTTGTTTGTGTGTTTTTGACTCAGCAGGGATATCCACACTCAGATCCCAACAGTTTATTGGCACAAAACGCTAGGGAGCTTGAGTTCCAGTATTCCAATTTTGCACAGTCTATGCAGTCAAGAAATAGCAATAATGCTTCTTCACTTGGTGGTCAAAGCATTTCCATGCCAGAGGTAAATAACCACTTTTGTCTTCTTTTTTTTTAAGAAACACAAGATGTCTTGTTAATTAGGTTTTGCTCGACTATGGAGTGATCTATATGTATCCAAATCTATACAACAAGAGGAATTTATATGATTTTGATTATATATTTTCTTACATTGTAGGCGCCCCGAGGCAGTGGAATCCAAGCGACGCAGCAAAACTTACAAGGTGCTAATATCGCCACTGGACCAGCACTTCCTCAACAGCTT
BG1288(SEQ?ID?NO：58)
ATAAGCATACCAATGAAGATATCAAAGAATGCAATATGTATGTTTTGTGTTGTGAAAGCTAAAGGATTCTACTTTATTTGTGTTGAGTGATGGTTCTTTAGTTTGGTGTTAATGTCTTGTGAATTGTGTTTGGCAGGTACAAGCTAGGTTTTTGTCCCAACGGTCCTGATTGTCGGTACAGGCACGCGAAGCTGCCTGGACCGCCGCCTCCAGTTGAGGAAGTTCTTCAGAAGATACAGCAGCTGACTTCGTATAATTACGGGCCTAATA GATTCTATCAGCCACGG AACGCTGCTCCGCAGTTGGGAGATAGTAATAAGCCTCAGGTGCAAGTTCAGACGCAAGAGGCGGGTAACTTGC[AGCAGCAGCAGCAGCAGCAGCAGC]AACAACCTCAGCAGTCACAACATCAGGTCAGCCAGACTCAGACACAAAACACTGCTGACCAAACGTCTCATCCTT TGCCTCGTGGGGTAAAT AGGTGTGTTCAGAGTTTCTAAAGTTTTTAATTGGGTTGTGTAAACTATGCTTCTGTATATCTGTCAAGACATTGTTTATTG
BG1321(SEQ?ID?NO：59)
ATGAAGATTGATGTATATTCGAATTTATAAAGTCTACGTTTAGTAAAGGTATACAAATCAGAGGTCTGAATTTGTTCAACTTCCTCCTCATTCCCCCATCCCCAAAAGAATCCGAGTTTTTTTGGATCAAGCCTATATAGATCCAAAAACCAACATAATGGCCCATTAAAGA TGCATAGACTCGAACCAAAC CGGATTAATACACTGCGGGTGAAACCGGTTTGGGAATTTTCACAATTGACTGAAGAATCAGGGTTTAAGGAGAAGTCACAGACCCAGG[AAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGCAGAAG]AATGGAGTCAGAGCACCAAACGATGGAACAGTTCCTACGATGGG CAGCAGAGCTT GGCGTATCAGATTCCATCGATCCTTCTCGATCTCAAGATTCATGTCTCGGCCATTCCCTTTCCGTCGCCGACTTCCCTCTCGCCGGCGGGTGCGTAGAAACAAAAATCACATCTTTTTATCATTCAAATTCCTAAACTTTTTCGACCATTGATGGGAAACTAGGAGAGGGTTGGGGGCTGTT
BG1368(SEQ?ID?NO：60)
CTTCAAGTTTCTATTATTATCAGTTCAGGAAGTGACACTCACTAGACCAACAGAAGAAGAAAAAAATCAACATACAGAAAACAGATAAGCACTGCTCATATTAATCATGAATCGTTCAACAAATTTGATCCGAACATTACAGAAACTATACGTGTTTGATCCAACAACGAAAGGAGCACAAACAAAATGAGATCAATACGATCGTTCTTCATTGTCGTTCTATTACAAAACTGTGCTTGCTTTGTTGGTTCGAACTCGAACATACAACAACATAGATAGTTATGTCGGGATATACTTATTTATATTTAGATTTAATTATGGATAACGACGGCGAGAGATTCTCGGCGACGGAATATCAACTGTTTCGCGATGAATGCTTCGATCGTTTTCTGAAACTCTTCGTTTGTCAGATTATCCTCCGGAAACGGCACTTCTTCGCACGACG CGACGGTTTCACGGCACTTCTCCGTCTCCGATCGCCGGAGCGTAGGTTTCGTCACCGTCTCCGGACTCTGTTTCGCAGAGATTTCCGTTTTGCTTCTTTTATAGACCTT[CGTCGTC GTCGTCGT]CGGAGGATGATCATTCGTCGATTC
BG1392(SEQ?ID?NO：61)
CTCAATCTTTGTGTGGGTGGAGCAATACATAAATTTTGTGTGGTTGGAAGGTTATGAAATGAAACTTTTAGTGGAAACGTGAATAGATCATCCAACTTATTTTGTGTGTTTTAAGAAAGATTAGATGAATTTGACTCAGCTCATTGGAGAGAGAGAGAGAGAGAGAGAGAGAGGCTTCACAGAGCCCATCGAATCCTATGCGCGTGTGAAAAGCACGATCCAATCACGAAGCTAAAATCTTCAGCTTCGTTGTATAAAAAAAACTTATTGAAACAAACCTCAAATTCCAATTACACCCTTGACAGCGATACACACTCTCTCTCTCTCCAACTAAAACATATCTGGAAATTATAAATAAAATTTATACTTTATCTGGTAACCCATCAAATAAAGCTATTAGTCACATAATAGATGACAAAAAAAAAACAAATAAAGAAAATTTAGGAAACAAATCTACTGAGATTAGGCTGTAAATCATACGTATATCTTTCCCGTATACAGAGTGCCGTTTTAAGTATAATGTCGACACGTGTCGGTCAGAG GCTCGGCTTCCAAGGGTAAGATTGTAAAATCACGATCGTCATCTCTCTTTAAGAATTTCCAGAGTGCT[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]GGTGCTTTCCCATAGCCATTCACGTC[GAGAGAGAGAGAGAGAGA]GGAAGGAGATGGAGGATATACAGGAGGAAGAGAACGGTACGGACGAGGAGGTGCTGGGATCGAGCTTGACCATGGAGAAAGT GGCGGCAGCTAAGCAGTACATCGAGAATCACTACAAAGCTCAGAATAAGAACATTCAGGAGAGGAAAGAGAGGTATTATAAATCGTCTCTTTCGTTGAGTGAGAGATTTGAGATTTGGAATATCGTTTTTTTTTAGAGACTAGTTAGGGCGAAATTAGTTGCGTGAGCTTTGATTAGTCTCTCGTATTTGATGATAATCATGGT
BG1442(SEQ?ID?NO：62)
GAACAATAACCTGATCAAGGCTCTGCTCAGCTGCTTTACGCTCGTCGGGATTGGGACTGCAAGCAGCCGCAGCGATGATTACTGCCAGGTTAGACAGATCCATCGGGAAAATTCAGCAACAGATTCTCCGAGGAATAGTCGGCGTGTCAAGAATTTTCCGGTGAATCGAAGAGG CGAGGAGGAAGAAG ATGACCGACGAAGACGAGGACGAG[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]GGTATAAGAAGGAAGGTCTAAGCTAGGGTTTTAATGTATGGTTCGTTGGTCTGTTATGGTAAGTCTATCGCACGCACGCGCGTGGTAAAAAAGTGAAAAAAAAGAAAAATCGACGTGAGACACGATACACAACCCAGACTTGCCTGGACCTCTAGTCACCTA TTTATTTTCACCGCCTGC TCGTTTAGTTAGTTACGGTCAATCGATTGACTTTTGGTTATTTTCTATGTGTTTTCAATATAATCAAATTCAAATGATTTTTTAA?TCAAATCAAATGTAAATAATAAATAATAAAAATCCAAATGGAATTAATTAGTTTAAACTTATTAAACTATTTTGTCATCTTTTTAGTTATTTAAGAATTATATTAAAACTTGTAAAATTCTAGTTACAAATATAATTTAATGCATAAATCTAATGAATTTGGGAGAAAATAG
BG1449(SEQ?ID?NO：63)GGTTTCCGCCGCAATGATTTGGT AGTTGTGGTGAACAGGCTGCATATGTTTATTTATTAAGGTCCATAAGATCATAGAAACAATGGTGAAA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]TTCATACAGTGGTGTTGGCGG AGACTGGGATGGAACGAGTTGGGGTGCCAATTTCTGGGAAGAGGTAGGTGGAGTGGTTATCATCATCACTGGATGAGTAGTATGCTCCTCCATGAAGTGGACTCCGAGGAGGGCTGTCCTGCCGCATAACGTAGCCATCTGGCTCATACATTGCTTCCATTTGCTCATCATTATGATTGTTGCTGCGACGGTCAGGCTGCGATTTGTTTAGGAGAGCTGATTATTTATTGTCAAGAATATGTTTTATCACTAGAGAGAAGCTCAGGCTTGAGAATGTTGTTTGAGTACCGTTCTGTATTGTTGTGGTGGTGGCGAACGGGTCGCAGCTGGATGAAATGGGCTTGGTGGAGAGCTGTTTCGTGGTGTTACTTCGCCGTCTCGCTCATACACTTCCTCGGATTGCTGATTATCAGGGTGGTTTCTACGACGATCAGGCTGTTAGAATTAGAATAACGAACTTTATTGTCAGGTTAAGTGAATCACTTTATCTGTTTCTGGAAAATAAGGCTGAGTTTTATGGTACCGTTCTGTATGGTTGTGGTGGTGGTGGCGAACGTCTTGCGGCTGGATGAAAGGGGCTCGGTGGAGAGCTGGTTCGTGGTGTTACCTCGCCG
BG1453(SEQ?ID?NO：64)
AGTGCTTTAGGAGAGGAGGATCTTGATACAAAATTGTTCTCAAGAATTGAGAAGAGGAAGAAGAAAAAAGGGTATTAAAAGGAAGAAGAGGAGGGCTTTATTAATCATGTATTGCTAAAGAGGAAGATGAGGAACTCA TC ACCTTCCTTCCTTCAATGGCAATGAAATGAAAA[GAGAGAGATATGAATGAGAGA]GTGAGTGAAAAA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]GTTGGGTCTACCAT GAAAA GCAGAGGAGGTGGGTCAAAGCAAAAGTTTTCGACTCTTTTTTTAGGGCTTTTCAATTTCCTTTTTTTCTTTACTTTCCATGTTTGTATATTTCCCAAAACTCGAATTCATACACAAGTATCTCGGGAAAACGGCTTATTCATGCCCGAACTAGGGGTTGCTGAGAGAATACATACCTCAACTTTTACTGCAAGTCGAAACATAC
BG1513(SEQ?ID?NO：65)
ATAGGTTCTGTGTCGTAACCATTAGAGTCTTAAAACCGTCAATTTCGATTCTCACATCCAAAAGGTTTTAACATTTATATATATAGTAAGATGGGTAAAGTTTGTAACCGCTCGTGTATAAAAATCAGGTTTACGAAAATCGCATTACTTTTGATTTTCTGATCGAAACAGAGCTTCGAAAAGGAACTACTACGCAGTAAATAAACTTACTTGAAGAAACGAAACTTACTTCGAAAAGGAATTACTTTC TGAAGGTTGCGATAGCGAAGAGAAACTT[GAGAGAGAGAGAGAGAGA]GCGAGCGAGAGAGATATGAGAGTGATACCGCGGCGGAAATGAAGAAAAAAAAAAAAGGTTTAGGGTTTAACGACGACTGTTGCAAGTTGTAACCTTTGACTTGTTTTTTTTTAATAAATCTTTTTTTCTTTAAATTAAAAGAATAAACTCTAGAGTGGAAACCCCCTGATAAGTAATAATGTTTCAGTTCCGACCCCAAAGTAAAGTTTCAAATAATTTAACCCAACTTTATTGTAAATAAAAAAAATATTTCGATGAAACGAATATGTGCAAAATTTCATATATCCATAATTCATTACGATGTGTTTTATCAAAAAAAAAATCTTTT
CA0105(SEQ?ID?NO：66)
GGTTAGTCATCCAAGAACAGAAAAATCTCTAAAGAAAACAAACAAACCTTGATGTAATAAGCCTTGCGAATCTCTTCCTCAGAAGCGGAGGGAGTGACACCAAGAACATCATAATATAC TGTTTCCTTCACCATGATCGGATCAAAGCAAGAGATGTAACTTTTTTGTGTAGATAAGAAAATAAAGGTTGGTTTTGTGGATTGTGTGTGAAGCTTTTAGGAGATATGGG[GAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAA]GTT GGGACACAAGAGAGG TAGGGTGTGTGATCTGCTTGAAGAAGCAAATAAAGAGATGTCTTTACAGTTATGCACTTTTGATTTAATAAATAAAAAACTTTATAGCGGGGAGGCTACACTACACTTTCACCATCTCTTTTTAACTGTCCACTCAATTGCTTTATTGATCTCATGCCTCCTTTTTTATTATTCCATTGCTTTTCTGTATTGTTGAACATACTGAAGAAAAAGAAGATGATGATTTTTGCAATACGATTGTGATCTGGTGTATCTTATCATTTAGCCAAATGGATATTAAGTTGGAAAAAAATTCAAAAATATAGGTCCTACCGGGAGTCGAACCCAGGTCGCTGGATTCAAAGTCCAGAGTGCTAACCACTACACCATAGAACCTTGTTGTCTTAACTTTACTTTATTTATTTATAATGAAACATTATAT
CA0120(SEQ?ID?NO：67)
TTTAAGCCAAAATGTCATAACACAACAAAATGAGACAATAATAACATTACTGTAACAAATACATAGTTTCTAATTAGAACAAA GACTAAACCA GACCAAGAGAAAAGTCGACAACAACTTTTAACTCTGTCCTT[CCACCATCATCATCATCATCATCATCATCATCATCATCATCATCA]TCCTCATAACTTATTGTTGTACCAGAACACACCTTTCTTCT CACCTTGCCTATCCGGTTC AACATAGATACACTCCTTCGCCTCCCTCCACATCGCCTTAACCACCGGCGTTCCATCAAACTGGTAATACTCTCCAAGTATCGGCTTTATCGCCTTGGTCGCTTCCATCGCGTTATAATGCGGCATCGTCGAGAACAGATGATGCGCCACGTGCGTGTCCGTGATGTTATGAAACACCTTGTTCAAGATTCCATAGTCTCTATCCACAGTAGCCAAAGCTCCTCTCAACCAATCCCACTCCGAAGAATATAGTGAGGCAGCGAAGGGTGCGTGTGCTGCAAGTAAGTGATCAAGACGAGGAAACAGTTGACAATCATAAGCGGAACTCCGTAGACACAGACCATCGAGGCCACTCCTCGCGAACCAGCGTAGCGGTAGAGACCGTAACATACGGAGAGGACGCCAGCGTCAGAGATGTATATCTGGAGACGCTCGCGGTCGTTGTAGATGGGAGCGTTCGGGTGGAAATGGCAAGCGAAACCGTCGCTGTAAGGTCTTCCAGAGACGTTGAAGGCTAAGTACAACGGCCAGCCGAGCGTGAACTGGACGGTTAGCATCACCGTGCGTCCTAGCGGG
CA0163(SEQ?ID?NO：68)
TGAGCTATTAAAACTACATTATCTTAACGTCATATCAATTTGACATTTGCTTAATATTCATTTTCTTAGAACCGCTTGTCAAAAGCTTTCCCAGTTTTCCATTAACTAAAGAATGCTATCAGGAGAAGTTTCTGAAATTAGATCATCATCATCATCATTTCTTAGCAACTTTTCTGAGATTGAGATCATATATTATCATCA CCATCATCACCACCACCATCATCATAATCATATTTGCTTAGCAAATTTTTCTAAGAATCGTATATTATAACCACAAAATCTATATTTACTAACTTACAAGATAGATCCCATAAATTTATAACATTCTGCGATTACTCATTCCCTATATAAATAACGTTCCATCTATTATATCCTACATTAT[CATCATCATCATCAT]CACCATCACAAT[CATCATCAT]CACCATCACAATCATCACCATCACAGTCATCATTTTTTCATAGCAAACTTACAATTCGAAGAAACGAGCGCCAAA ACATCCGACCTTCTCCAGCAAGACTGAATCCAAAAATCCGAAATCGACAACATCTCCAGCTCATCACGAACCCTAGGCAGCCACACCCGCACGAATTCGACATCGGTAAGGTACAATTCGTCGGAATCGTCTTCGACCAAGTCGTCGAACGACATCCCGCTGAGCGCCTTATCAACCCCAACCGATATGACCCTAACGTTGCGAGAATCTTCGATCAGATTCCTGAAGACCGTCTTGAACGGCGTGACGGAGGTCGAGCGCGATTTGGAGTACCGTGACAACGTGCATAGG
CA0221(SEQ?ID?NO：69)
TGTTCCTCTGTTTCTTCAACCTCAAAAGCTCTTTCCACAGAACAGTGCAGTTCGGTGGACGA GGAGAAGGGTCGTCGTCAAGAAACCAGCTTCCTCTGTTATTATTCTCCTGCTCCTTGGTGCTGTTGTGAACAGTCTCCTCTTTCTTAACTCTACGAT[CTTCTTCTTCTTCTTCTTCTTCTT]CTACTACTTCTACTTCGTCTACGTCGACGACTTTTGTTTTCAAGGTAACGTTTTGAAGCTTCTCCGAGTGTTTGGCTTGCCAGAAAGGAAGCAGCTTTCCTTCGGAGAACAGCTCGTCTGCGGCGG TGAGCATCGTTTGTGTGTTCGACAGAAACTCAAAGTCTCCAGCTTTCACTTGTTCTTCTTTTCCCCTTAGGAGATTCTCAGGGTTGATGCAGATGTAGTCTCCCTCGCTGTCTGATGATGACAGATCGGCGGAGAAGGAAATGCGAGGTCCTTCCGTCGTGAAAACCATCGTAGCCTCCGCCGTTTCCGCTACTACCATGATCGTACAAATGTGTAGTTATGAAGTGAAAGACAAATCAAGTGGA
CA0226(SEQ?ID?NO：70)
TCTTAAATTTAAATTCTATTCCTCAAACACTAAATCTTAAATCTACACTCTATATCTAATACTCTATACCACAAATTTAAACTCTATATACAAAATCATAAACTCAATCTTTACAAACTTATAATAGAAACTTTAAATTTAAACCTAAGTATATTATAAAACTCAAATTATATACTTAATCCTAAATCTTAACCCTAACTCATAAACCACATATTTCATAAAATATTAAATCTTAATTTTTAAATAAATTATAGTTCCATAAATAAATTAAAATTTCAAATAAAAAATTTAGATTTTAAATTTGAAATTATGATATCAAAGTATTTAAAACTTAAATAATTTTTATAATAGCTATAAATAAATAAGAAGATAATTTGTATTGTTTTTTTATACCATTGATTGATTTGAATCAAAGACTCAAGATAGCTCTTGTATCTATTTTCGCCTTTTTTTCTTATCGGTAGTTGTTGTTTATGG CATGGATCACCT GCACCCTTAGATAATATTGAACCAGAGATTAATTGTTCTTTTATTCTTTTTTTTTTAATTTACTTTTCTCAGATCTACGAAA[GAGAGAGAGAGAAGAGA]TGGAGTTCAAGGTAGAGAAGGAGAACGCGACGGCTGTTCGTC[ACCACCACCACCACCACCACCACCACC]ATCGTTCGTCACTACCACCTTCGCTTCTCA GATACGTCTTCACCGGAGTCGCCAGAACCACCGTCACGCTCGTCATAACCAACATCGCTCGTTCCCACAACCACCGCCATCTCTCCCATCAGCCATCGCTCGTCACCACCACATCACTTCTCAGATCCGTCTTCACCGGAGTCGCCACCACAACCGTCACGCTCCTCATAA
CA0233(SEQ?ID?NO：71)
TTTTTTTGGTTCTTTTAACTTTTAATAATTAATTCAATAATCTGTAACCTCTCTATGTATCTCTTCCTCATTGTATCTGATTGAGTTTGAATCAGTTTTTGAGCAGGACGTGGTGATGCCAGAAGATCTAGCGAATGTCCTTAGGACAGCGAAAGAGATTGTCGTTGCCACAGTCCTTCCCGTCACACTTTGCTTTTTGTACATCTCTATGCCTTGACGCCGCCGTAGGAACACGCTATCAAAGCTTGCGGGGTTGGAGGGAGCTCGACGACGGAGGA GGCAAGCATGGT CTCGTCAGATACAACAGAGTCAGCATTATCTCAACATTCCGGTCATGTAAGCGGTGTAGTCGTTTATACAAATTTGATTTTCAGATCTGAGATTCGCTTTTTGACTTTACAGTTTTTGTGTATATTTTTGTAGGATAGGATGAAGGG[GAAGAAGAAGAA]GGAGGAGACGAAGACGAGAGGCTGTGTGGTTGGCGTCAGTTATAT[CACCACCACCACCACCACCACCAC]TACCGTCACGCTTGTCACCACCATGCTCCTTTCCATTC GAGGCGGCTGGAATCTTTTTTTTTCTAGGTTTAGA
CA0328(SEQ?ID?NO：72)
ATGGTACAAACAATCATACAGACACGCATTCTCTTCTAGTTCAGCTGCTGCTTTCTCTAAGCTTGTCTTCATAAAATTGTTACAA ACTTTGGAG CATCCTTCCTTGGGCCATCAAGGGACCGATACATGTACAACGTCTCCACATAATCTGCATTGT[CGTCTTCGTCGTCGTCGTCGTCTTCGTCGTCGTCGTCATCATCAT]CTGACCCAAACACCT[CCACCACCA]CACTAGGCAGAGTATGAGCAACATCCCTGCAGGCTCCCCGGGACAACCTACATGACGACATCCAAACAAACCTCATCTCGTTATACCTATGCA TACCAGAGCGCAGTC CAACATCACCAAAGGGACTGTCCCTGATCTCAAGCTTCTCTAGTTTAGGACACCCCTCGAGGATATATCTCAGACCCATGTCACTGTCCCCTGCAAAAGCTACAGATAGAGTACGTATCAGTTTCCCATACTCTCCTATAAGGCTAAAGGCTTGGTCCGTTAGTAATCCAGATACTGCAA?GCCTGGTTAGCTTCTTGCAGTTTTTAACAATGGCGCCAAATCCATCGTCCATTGGCTTCCTTGTCACGTGGTCAGGCCTATGGCGACCCATTATGCAAAGCCTAAACACGGTAAGCTGGGGACAGTTCTCAGACATGGCTGTCACAGCTACATTTGTCATCCGCTGGCAGAAGTAGAGAATAGACTCAAGTTTCTTACAACCTTTCTGAAATTGCTTGGAGGCCTAATCCCGAGACAGGACCTTCACTGTCTTCACTAGGATCCAAAGGGAAAATCCCTAGCTCACGGAGCTCCTTGCATG
CA0410(SEQ?ID?NO：73)
CGCCGCGTCTCCTCACTCTCCGGATTACTCTCCGTCTGAATCCTCTCCTTCTCGCTCGCGATCTCCCTCTCCTCCTTCCCGCGACGCTCCCTACCGTCTCCGATCGAAAGCCGCCGCCGCCTCCGCGAATCAAGGAGCTGGTGGTAATCCATCGGGAAGCCGTACTACTAGGAGCCGTCAACAAGCTGGGAACATCCGTACGTTCGCCGATCTGAACCGTTCCCCCGCTGACGGCGCGGATAGTGATTCCGACGAAGGCCAAGAGTACTATACTGGTGGACAGAGGAGGTAAAATTGTGTTTATATTGAATGATCATAAACTGAGTAATGTGGAATCATGGAGAATTGTGCTATTGATTGTTTGTGTTGGCTTCTCTTTAGCTAATGGATTGGGCCTTGTGTGTTTAGTGGGATGATGGTTCAAGATCCTACTAAGAAAGCAAAAGATGTTGATGCACTCTTTGAGCAAGCTAGGCTTTCAGCTGTGGACAGGC CTGTTGAGCCATCGAGATCAGCTTCTACAAGCTTCACTGGAGCTTCTAAGATGTTATCTGGTGAGCCTGTTCCCTCTGCTACTCCT[CAGCAGCAGCAGCAGCAG]CAAGACCAGCCTCAGTTGG TTATGCACAC CATCACTTTCTGG
CA0423(SEQ?ID?NO：74)
CCCTATTTCGCTGAATCTGCTTTCTAACCCTAATTTTCTCGATTTTTCTGCTCAAGCGTGTTGGCAATGTCGGAGGACATGGTGATGCATTTCTCCTCCAATTCCTCC AATCAGTCCGATCACTCCCTGCCCGACAAAATCGCGAAGCTCGAGGCTCGCTTGACCGGCAAAACCGCCTCCTCCGCCAAGCCGCAGCCTC[AGCAGCAGCAGCAGC]TCTCCGTCTGGTCATCTGCTTCCGCCCCTGCCAAAGTCGCGGCGGGTTCGTCGGATGTCTCTATCAGTGA TTC CGACGACGAGGTAACTTCCGATGATTTTTTTTTATTATTTTTTTTTTTAAGATTTGATGTCTAATAGTATTCTCGTTGTTACTACTGTCTCAGAACACAGGAGATTTCCTGATCCGAGCAAATACCAAGAAGCGCCAGAAAGTTCAAGACTTTAACAACAACAACTCCACTCTTGTTGATCATGCTGAGGTAGTGAATTTTCAGTTTAAATATCGATCTTTTCGTCCCTTGCCTGGTTCGTAGTTATATTGATATGGTAACTAAGGTTGTGCGATACTGAAACAATCTGATATGATGCAAGTTTTGTATTCCCTTTTGATGAATTATTATAATGTCGAAATTGAAGCCGCAAGAGGCAGCATATGATGGAAGGAAAAACGACGCTGAGAACCAGACAGGCGTCGATGTGAGTAAGAAGAAGCAAGGTCGAGGTCGAGGTTCATC
CA0456(SEQ?ID?NO：75)
C TTCTGTTAGAATTCTACCG[TTGTTGTTGTTGTTGTTGTTG]TCTTGGTTGTCTTAGAAGC[TCAATCAA]CGCCTCCGCCTTTAGCTTAGCTCGACTCTTACTACTCTGCGACAAAG[CCACCA]CAGGAGCAATCGCACCTTCTCGCGCCACCATGGTTCGATAC ACCACACT[CTCCTC]ACAAAGCTGCAGCAATATCGACACGCCCATCTCCTTCTGCCTCTGCGTTCCCACCTCCACTATCTCCACAAGCACCGGAACTCCTCCTTCCTCCACCACCGCCGGCTTCGACTCCGGCGCCGACATCAGCAGATTCATCACGTACGCCGATTTATCCACCATGTTCGAATCGAAATCCGCCATCAGCTCCACGAGCGGCTTCATAACTCCCGATTCCACGGCCCTGGTCTTGTTCTCCTTGGCCGAGCAGAGCGAGTAAAGAGCCGTCGCCGCGTCCTTCTTCCCCCTGAACCCGCCGGTTTCCAGAAGGTTCACCAAGTGAGGAATCGCTCCGGATCTCCCGATCGCGATCTTGTTGTCTTCGATCTGCGATAGGCGGAGGAGAGCGCAGGCGGCGTTCTCTTTCGCCGTCGGCGTTCCCGATTTCAAAACCCTAACGAGCGGTTTAATCGCGCCGGAGGAAGCGATCAGCTCCTTGGTCTCGTCGCAGAGGGAGAGGTTCAGCACAGCGGTG
CA0488(SEQ?ID?NO：76)
GCCAAGCTCTCCGACTTGTCACCGGTGCCAAGAAGCAGCCACTGAGAAAATAAAACTCATTCAAGATTCAAAATCCTTGTGTGCTTCTTCAATGCCATTTTAGTTTGACTTCTTCATTTGCTACAGTTCATTAGTTATTTCCTTATTTGCAAAAGAGCCCTCGAGTTTGTTAGAAACGTGAAATAAAGCCATTAAATACCAATTCCCTCCACTTTGAAGGGGTTTTGAATATCTTTCCCTCGACTCCAAAATCCTCGCCGGCGATAAGCAAACCCTAGATTCGATTCGCCGTCTGTTCATC CAGCAATGTCGTCGTTCAATCCATTCTCTACCCCACAGCGACATCAGCAGACGCCTCAGCCGCAGAGCATCTCCTTCTTCTCGCCACCGCAGAGCACTCC[CTTCTT]CTCTCAACTG[CAACAA]CAGCAAACGCCGTCGTTTCAGCCGCACCAGTTCCAGCAG[CAACAACAACAACAACAACAA]AGTCAGCAGCAGCTGTATTTGTTCACGAACG ATCAAGCTCCGGC GAGTTACAGCACCGAATGGGAGTGATCTTTCATCCGATTCTCAGAAACTTCTCCTTGAGATTGAGTATTGCTCTTCTTTC
CA0546(SEQ?ID?NO：77)
ATCAAACCATAGAAATACATGACTTACTACATTCACTCATCTGTGTTGGAAAACTTTTCAAATTTATTATATCTTAATTTATATTATTTACAAATGTTTATAATTGCATGATTTCAATTATCCCCCATCACAACATATTTTAAAAAATTTAAAAATTATTTTTAAGATATACAATATGAGAAGATTTTCAGAAGGCTTCTATGAGTATGTTCTTAAAAATACATTCTATTTTTTTTTTTTGGTCTAATGGACTATTTATAATTTCAGTAGCATTTTAGATTAATTTTGCATTTGATCCATGAGGTATATCTTTGTGTTTAAAACCAAGTTTTAGGTTATATTTGGAAATTTCCTCTTGATAGTTTGAAGGTTTGAAGTTTTGATGCGGATAGCAATGGATAATAAAACGGATTTTGGATCTAGGACAATAATTCGTCCATCTCCTACGTGGGGTCTTTAGTGATAATGAAAAAACTCTTCTGGTAAAAACAAAATGTTTTAATAAATATGGGGCTCATCCATAAGTGAAAAATACCTCTCTTCTTCACTGCAAATGAATTATAAA CCCCTTCCTTATCCACACACA CACAGACTTGTTCGCTCTCTTAAACCCCT[GAAGAGGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAA]GGCGAATCATGCAGATTTGCCAAGCAGCGGT AACCTTCACCTTCACGAACCCAACAAACCCTAATTTCTGCAAACCCAAACCTCTCTTCCCAAGCTTCCAACCCCCTCGCCGCGTCGCCTTGCCGCCATGCCGTGGCTTCAGCTCCGACGAGTTCCCCGTCGACGAAACCTTCCTCGAGAAATTCGGACCCCAGGACAAAGACACAGAGGACGAAGC
CA0552(SEQ?ID?NO：78)
GATTTAAAATGACAATTTTTTATTGGTTGGTTC CGCTAGGGGGTGA ACCAAGAATAACT[CTTCTT]TTATTTCTACGTTCTCATTCTTT[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]CTACGATTTTTAATTTCATTCAATGAAACAACAAAGTAGATCTGATTTTTATTTGAGTTTGGGTCCAAGAAGTGAAGAAAAA TATTGGAGAGGAGGATCGACGCCTCTGTTCAATAGCCATGGAAACTCTGTACGCTTCCTCTCAAGCTCCGTGGAGAAGAATACCAGGAAGACGGCGAGCAAGCTCCGTGGAGAAGAAGACCACGAAGACGGCGAGCGACTATGAAGGTGGCTGGATGCAACGACAAGTACGCCTCATCTCTGAAGCTTGACTCTCAAATCCCACAGCGAAGAAGAAGAATCAAGGCGGATGTGAAGAGCATAGAAGAGACAACGACGAAGGTGGTGGTGGGGCTGGTGAGGACTGGAAGATGAAGAAGACCACGTAGGTGGTGCCATGGTGGTGTGCGGCGGCGTACAAGATGAAGAAGACGACATATGGTAGTTAATTAGAAATTAGGTTTAAGCTTGGTTTAGGGTTTTGGTTTATTTGGTTTGGTTTTAGTACTTTTTTATCTAATTAGATTTTTTTTAATATTTTTGTAAAACAATTAA
CA0603(SEQ?ID?NO：79)
CATCATCATCCATTCATCATCATCATCATCATCATCATCATCATCAGCCACCTTTCTACTTTGTCTGTTTCACAACTGCCCAATCTA CCTCCCAA AGTCGTCTCTTCCATGTGATACACTTCGCTTGGCTTCTTCTGCCTTTAGCTGTTCCCGTCTCAACGTTCGTACGTTAAAGGTAAAGT[CTTCTTCTTCTTCTTCTTCTTCCCTT]ACGTATTTTCGTTTTCCATCTAAAGATTCATTCTCTCCT CCGAGTTTCGTCCCCTGTCTACTCTGTTTCTGTGATGTTGACCTCTCTCTTAAGCTGATCTGATATGTGTTCTTCTTCCTCTTTGATGCTTCTGTCTCTGTAATTCTTTGACTACTTTAGATATTTTATCTTATGGGTTTCATTAAACTCGCACAAAGCTCGTGACTTTGAGTTATATAACCAGTTCAGCTCTATTAAAGTTTTCTTGTAGACCAAACACTCATGAGTTACAGTGTCTTGTTCTTAATCTTCCTTTTGACTATTTTATGAAAAGTTCTTGATCTTCGTTACTTTTCAATAGTCTGATTC
CA0614(SEQ?ID?NO：80)
CGAGGGACACGAGGATAGGACCTGGGATGCCTTGAGTGCAGGCTGTGCAGCACCATAGGCGGGCACCTTTTTAATATTGTATGTTGTAATATTTCATCTAAAATTATAAGATAATAGGGTATACATAACTTATTTGCGTGTAAATAGATCTCATTTCTACATTTGAGAATCATGAAAAATATATATG TTCC AAGTGGTTCTGCAATGTGTTAAA[TATATATATATATATATATATATATA]GATATTATTTTCAATTAATATACTCACAAAGTTGGTTATGACTTATAGTACAAAACAAAATGTGGAGTTCATTAACTACACGAAACCCATTTGTCCACAATATTGAAGTAGTCTTTTGTGATGATTGACATAAATTCTCATTTTAA TTGCCCTTTATTGGGATAGCTGACAACAACAAAATAAGTAATTCTTTTCAGATTTGAGAAAATTTCAACTACATATGTAAACAATTCAAAGAACATAAATATAATAAGAAATGTGCACAAAAAAAAATATAGAGATATATAAGAAATAGGTAAATGAGGCCAAAGATTGTTGTTATATAGAAAGCAAGTCACTGCATCATAATATCATGTGGTGGTTCAACTTTATGACGATAGTGAATAGGTCCTCTCTTACGCCTTGAGATTTTGTTTCCGTTGAAGCTGCAGATAACACACCTACACCTATATCTTGGATGATAGTGATGATGATAATTATGATGATGAGGATGATGACGCAAGTGATGATGGTTTTTCCTAAGAGCGCAACAAACCGAGCTGTCTGATACC
CA0636(SEQ?ID?NO：81)
CTCGGAAGAACGAATCGGGTCAGCTCAATGCTCCATCGGGTCAACCTTATCTCTACTCGGATCTCTCTCTCGGGTCGAACTTCACTTGCGCGACGCGAAGCTCGAACAGCTCTGTGACTTGCTGGGGAGGAGGAGCGGAGAGGTTCAACAATGTAACCGAAAAGATCTCATTCGAGTCAGTTACATCCGGGTCGGGTCTAATCTGCGGGTTGATATCCGGTAACCTCTCGGTCATGTGTTGGAGCCCTAATAACTTCTCAAGAATCTTCCTTCCTTTCCCAGATATCTTA CCAGGTCCTTGCGTTGAATCATCTATTTGCAAATGTGGTGTGTATCCACGATCTGATCAGCTATGCTCCGGCTCGGGTTCGATCTGCAGCAAATGCAAAATCTCA[CCTCCTCCACAACCACCATCACCACCACCACCACCA]CCGTCAGATTCATCTCCATCTCCATCTCCGCCGCCGTCGAAGGCGTTAACGAGAGGATTACTAGCGTTTGCGATCGTTGGATCAGTAGGAGCGT TTG CAGGGATATGCAGTGTGGTGTACTGTTTGTGGACCGGAGCTTTCTTGGGGAAAGAGAAAGTTCATAACTCGGTTCAACCGACGATAACCCGCGGCGGTTCGAGTACCCGGTCAAGCAGCTCGCCGCCTTCTCGGTCCTTGACGAATAGACGTCAGGGATCGAGAATATTTTCGATGAGAA
CA0681(SEQ?ID?NO：82)
TATACTGCTAACTATTTACATGTAAAATCTCTGTCAATTATCTTTTCCTATTCTATACAATTTTCCACAGTTATTTTTGTAGTTCTGTTGCTGAACTTGAAGCTGAGTTTGTGGTGAAGAAACAATAACACCAAACACAGCATATCACCTCCTTCCTTCTTCTAATGCATCTCTTCT CCTCTTCAACCCCACACT GGATGCGAACCTGTTGTTCTTGCTTCAATCGTTTGAGCTTGGATTTTTTCAATCTCCATCTCCCCTCCTCTTCCACCAACATTACTCTCTTCATCCCCATCCAATACCAACTTCTCAAACCTACACTGTTTTGTTGCAAAACACACAGTCTCAGCCACATCATTAAAAAAAAGGAAGAGG[AAGGAAGGAAGGAAGG]AAAGTTTTACATTTTCACATTCCCATAAGGGTAGGT GAGACGGCTGA TACATTCGCAACTCTGTTTGAGACACGTACAGCTGATGGTTGTTATCCACCACACGAGGGGCCTTTCCTTTCTTAGTTGCTTTCCACGGCTCCTCAGCACTCTGCTTTATAACAACCTCCGGAAACACGTTACTTCCAGAACCCGAAGTTGATCCACCTCCTCCATATACGTCACTCTGCTGATCTTGCATCTCTCTTCTAGATCGGTTCTGGACCATGTACCACCTTCGTATCCTCACAACCGGTCCTTCAGTCTCTGCACCAGACTCGGAATCAATCCCCGTCATCACAAAGCCTCACCACCATCCCCAGCCTTCAACGCGTACTGTGTCATACATCCAGAAGGAGCAAAAACAGTAGATTCCTCGTTACGCTATTATCAGAAGCAGTA
CA0719(SEQ?ID?NO：83)
GCTACATATGTCCCTAAAGAGTGAAACTAAAGCGAATTGCGAGGATTATTGAACAAGTTTCCTTCCAACTTTCTAACGAATCAGCCATCATAGTAGCTCGCAATCAACATTTAGTTTCTGGGAAGATGAACAAACACAAATTACCCAAGAACACGAGACACCCAGAACATAAACAAAATCAAATACATCATGAATCCGATTAAAAAGAACGAAGATGGAGCAAAGTACCTTTTCTCGATTCGACTTGGAGAGAAACTCGAACGAAAGGGAATAAAACCCGAGGAGTGACTTAATTGGGTCACATAATTTTGTTAACCGGAAAGTTA CCGAACC GGAATCATACAGCTCGTTGTGTAGT[GGTTGGTTGGTTGGTT]TTACAACTTCCACAGACTAAAAATGACATGAAAAATTAATCAATTATTTACCTGAAATGTACGATTAGCCAACAATTAGTTCTGTTATTCATAACAAAAGAAAACATTTTAATTACAGAGGTGAACGTATCCTAAAGAGAAATCTTTTTTTTCAAAACAATTAAACTTCCATTCATTAACATTAACCATCGCAAATACAAATCAAGGTCCAATCACACATATACGACTCAGACTCA GGATCTGACTGGTT CAAACGCAGC
CA0736(SEQ?ID?NO：84)
GCTTTTATCCCGGTTTAATAACTTTGAAATTCAATCCATCGACGTGGTCGGTTCATGGTCGAACCAATTACTGGACCCAACCCGACTATACAACCGGTTCATGGTCGAACCCGGTCCAACCATCGGGTCGGTCCGGTTTTAAAAACACTGCTCTAAATGGAAAAATATAGTCCTTTTTAACTATTGTTTAATTCAAAATCTGTTGACTATAGTGATGGATAATACAATTTATATGGGATTTGAATTTATGTATTAGATGTAAAAATTGAAAAAGAAAACATATTATATACGCTCTACGAGCTTTTTAAATAGATTTATTGGACCTAAGTATTTCATAAGTTTTGAAAACATGGGCTTACAAAACCTTTTTAACGATGTTCAAC CCGG GCTTAGACAAACTTTATGAGATCACGGCTAGGCCTTTGAGTGCTATTATTTTATTTTATTTATTTCTTGAATTTTAGGGATTAATAAATGTGAGAAGGAGTAGATAGTACATAATTAGAGATTGATGGAACAAATTGCAATAATTTAAAAGTAAAAGGATTTAAAATGCAAAAAAAAATATGAGGACACATGTCAACAAACCCTCCTTCTATATGTCATAAGAAGGGAAAAAATCAACTTTATATAT[ATAGATAGATAGATA]GATATGTATAATTTGGGATAAAAGTCGGTATAGCCG TACAGGCGTTTGTGCGATCAATCGGTCATCAACTAAACAAAATTTTAAAATGATTTTTTAAACAAAAAAAAATATTATTTAATATTTATTAAATAATTTGCAATTTTTAATAAAAAATAGTTTTCATATGGGATAAAATTTATCAATCTCATCTACTATATAAA
CA0739(SEQ?ID?NO：85)
GATCTTATCACCAAATTTTATATTGTCACGTTTAAGTATATCTTTCGTAGAAACATATTTTCCTCGAAATGAAACTATATGATATAATATTTTTTTTGTCTAAACATTTTTATACT AAAAACTGATAAGATTATTGTTGGTAACTACAATTATATTTACCTTGATAAATATATAAAG[ATATATATATATATATATATATATATGTATGTATGTATGTATGTATGTAT]ATATATCATCTTTTATTGAATTTGGATAATAGCAGATTAATTAATATTTTTTAGATAATGATAATATATAATTAAATTTTGATTTACTCAATTATTATT TATGCAAGTTTAACT TTTATTTTTGGGTGATTTATTATTGTATATGAATATATAAATATATTATGAATAAATATAATTATCTAATTATTAAGCATTATAAATATAATTATTCATTAAATGTAAAATGACTCTAATTACTCTAGTTTTTAATGCGATAGTTCAGATCAAAAATATCAATCAGAAACTAATAATATCACAATTTTATATTAGAGTATATTTGTTTAATTAACTAACTAATACAATCTGTGAATTTGTATCATTACCAGAAACAACCAATTGAGCAAGTCGGTTAAAAAGTTCATGCCATGTTTTAATTTTTGAGCTCATACATTTTTCATTTACTCAGGATTCAC
CA0753(SEQ?ID?NO：86)
GAGTATCAGCTCTACAACATCGTCCGGAAGCAATTGCAACTCTTCGCGATGCCTTTTCAGTGTTCTTGTTTTGAACGGAAACATCCTTCTCGTCCTCGTCGTCTCTAAATTTTATTGAATTTGATCAACAGATCTGATATATATATATAGATA CAGGCAGCTAAGGAATCTGGAAACACAAAAAAAAAAAAAAGAGAAAATTTCCTCTCCGTTTAAGTAAGATTTCCTTTTTTGAATTTAAACAGAATCGAAACATCAAATC[TAATAATAATAATAA]TACAAAT[ATACATACATACATAC]ATATTACCTCAGACTCAGGCAATGAACAAGCCTTTTCTAATCCTCAGGAATCATCCA[TCTCTCTCTCTCTCTCTCTC]GTTTTGTTTGTGAGCATCGATG[CGGCGGCGG]CGCTTAGACAAAGACA TCTCATC GGGACGCCTCTTTTACAACTCCTCCTCCTGTCTTCTTTTGGGCTTTCTGTAAGGCCCGACCCGGTTTCCCTTAACGCCGGTACGTCCTTAGTTCGCTTACCTCGACCAAACTGGCCCTATCCGAATTTATTCTCTTAACTTAGGATTATTATGCAATTTTCCTCTAAGAGGTTCAGTTCAGTACACAAGGTTCGCTAAGTCTAATCCAGCAACTTAGCAGTCTACTAGTAATCGCAGCATAATGAACATGTACCTACTGCCTCTGTACTTTGGTATCTGATAATCCATCCATACACTCCTTCA
CA0834(SEQ?ID?NO：87)
GCCATTTTCCTGATTCGAATCCAGATACTGCATAATAAATTTGAGAATAATAATCACTTTTTATTCACTGCGACATTGTAAGAGTGACTGTTATTCATAAGGCTGTTACTGTTAGGGTCGAAGGCAACTATTATTCTTTTTTTTGTTAAAGCCTTTATTCTTTCTTTTTCTTTTTGAATCTTTAGTTCGTAAATATTCTCTTTCATATTCATAAAAAATACACAACACAACATATGTATTACTATTAGAGGCATAACCATTAACATTGGATTTATTGAGGTTAGTAATTATTATGGTTGTTTGACAACAAAAAAAAAAGTAATTTTTTTTTGAGCAAACAAAAAGTAATTATCTGACAATAGTAGAAACTAA AAAATGCAACCATGCAAT ACGTGGTTTTATAATCATTCTATTGTTAAATATGATGAT[AATAATAATAAT]GAT[AATAATAATAATAATAATAATAATAATAATAATAATAATAAT]ATTACAGAATGTTGATGTAATAAACAAAAATAGTTTGTTAGCTAACGCCTCAGATCGATCAATGAGTAATTCATTCAGTTACCACATAAAGAAACAAATAAAAACTATGATAAAAAAGTTTTGACATCATTTTTTATTGACATGTCAATATGTGATAATACACTCTCT GCAGCAGTGACAACAAATACTACAA AC
CA0837(SEQ?ID?NO：88)
TTGGAGTCCTCCGACTTGGTCTTGATACAAGATTGTGAGGAAATCACTGTCCGTGTGTGGCATCAAACCATACACCTCCGATGGTTTCGGACATGGTGGATAACGGTTCATCCTTAGATAACATGTGTTTCGCACACAGGTTTTTTTGAAGAAACTTGATTTCCGTCCTGATTTCTCTGCAAGGACCTCTGCCAATGAATATGCCAGAGCCTCGGATTCTGAAGCAAAATTTTCCATTGTTGAGCTGCGATATAAAATGTACATATTATAACTAAGGTTAATTTATTATAGAGACAAATAAATCATGTTAAATAAATTAGGTGAAATAAT TGCGAAAG CCATGAACCTTTCTTGTTCCTTTTGTTTAATCCAAGCCATGAATTGTTCATTTGTTTTAACGTAGAATTGCTAAGATTTTTTTTTTTGTAAACCATGAATTGAAGTTATGATAAGAAAAGAAATGGAAAAT[ATTATTATTATTATTATTATTATTATT]TAATGGTAGAATGATATAGTATAAATAATTATTTCACGGTAATTATTTGATTTGGTAGAAAATTGCGGAAATTATTTGATTTGCTGAT TTTTTTTGTGAAGAACAAAATTCACCTAACAAAAGAAACACGTAAGATTATTTGTTATTTGTGGTACATAT
CA0896(SEQ?ID?NO：89)
TAAATTTACATGTAAATTACCACGAAACATTTTCTTTGTAACTTTACTACGACCTTACTACGAAATTCAGTTTTGTCGTAAAATCGTAGTAATTTTCTCGTAAATTTACGAGGAATATATTTCCTCGTAATTTTTCCTTGTTATAGGCATGTTTTCTTGTAGTGTTTATGTTGCCGTTGTTCTGACCACGATAGTTATGATACCTTTGTGATTTTCTGGTCACATATTCACTTAATTATTTTGTATGCTGACATAC CTCATGGGAGGTTCGCTTGATATAAATCATCACTTACAAACAAAAAATATTCATAAAAAAAAATATTCACACGTTTACAAAATCAAAAAGAGTTATATATAAATAGCTAT[AATAATAATAAT]GATACTAATATTAATAACAGT[AATAATAATAAT]GTTTAGAAAGCTAAACAACAAGGATTAGAACATGTATTTTTACAATTGCAAAAACAACAACAAAGTCGTAGCTTAGGACATTTAAAACAAGA TGACCATTTGATCTTGCAGTTGCAGCTGCAACATGAGCTCTTCTTATTAAGACATGATGGTCGACTGCAACTGCGGAAACATGTGGGTATGCAACAACATAAGTCCGGACAAGAACAACAGCAACCTAGGAAGCATGAACAGCTCGGGCAGCTCAGGCATTTGGGGCAGCTCGGTTTCGGGCAACAACAGCTGTTTGAGCAGCAGGACCCGTTGCAGCATTTGGAACAACGCAGCTACAACATGTGGAAGTGCAGCATTTTGGCTTCCTCAGATGGCACGAACACTCGGGTCGG
CA0991(SEQ?ID?NO：90)
GGCCGACGGCGATCTGTATCATCGCCCTGTGGTGGAATTCCTCCTCCGAGGGTATAGAAGGAAGCTAACTTGGTCTGCTTGTGAAGCTAAAATGAAAGGCTCGAATTTGTTGTACCTTCGTCCACCGTTGACATCAATAACACCGAATTTCTTAGACCGAACACCTCTGTTGACGACGGGGCAATGGAATGTATGAAAAACTTAAGGCAGTTTCAGGTTACGTTTGCAATGGATTATTCTCAATTGGTGAAGATGGTTTTCTGAACCAGATGAATGACCAGCATTTGAAAGCTACCTGGAAGATATTAAGCTTTTGCGACGAAGTTTCGTCAACTCAGATATTATTCATGTTCATAGGGCGGAGAACATAAGGGCGGATAGCTTGGCACACGTAGTACTCAGAAACAACCGTCTTTCGTCGTGCATATGGACGCAGAGTTGC CACATTGGTTTACAGAGTCTACATGAGTCTGTAAATATTTGCTGTTAA[AATAATAATAATAATAATAAT]ATATATCTGTCTATCAATTTTTAAAACACAATAAGTTTACGGTATATTTTTCATTGAATAGAT TGTT TTCAACTTTCACATGTATTTGTATCTTCTTCTATATATATATTTTCAGATTATTATTTCATTATTANAATCGTAACAATATGTATAAAAATTAGTAAAATATTGTTTTGTTGTCATATTCAAAGATA
CA1027(SEQ?ID?NO：91)
AGCGGCGATCTGATTCTCGCCCTGTGGTGGATTCTTCTTTCTTTAGTCTTTCCATTATTCTATGACGGTGTAATTCCCTATATATAAAAGGCTCCTTATATTTATGAATAATATAGAAACATAGATTTCATTACGACTATATTATTAGTATA TCAGTCTAGGCGTTTACCAATACCAATATACTTAATATATTTAGTATAATATCTTATGATTTACAATTATTTTCATATGATTTTGTACTATAATATGTCAATTATTATAATTTATAAAAAACTT[ATTCATTATTTATTATTATTATTATT]ATAAACCTACAACCTTTCAACTTAATTAGAATTCACAACCTTTAGAATTAATTGAGATTCTTATTATTAATAGATATTATAATCTTTTAAATGGTATATAAGATAATCACCACGGTACTAGAAAGCC TAGAGCCAAAGC ACCGCCTAAGCCGCCGCCTAGAACAATTACCTAATTTAAAGAAAAACTAATACTTATATTTGATTTTGAAATTTTATTAAACTTTGCAAAAAAAGAAGAAGATGGAAACATGTTAGAAACATATATCCAAATATAAAAATATAAGAATAATTTTATAAAAATTAATGATTAAAAACATATGCAAGATTTCGTATGAAAAAACTATTCTGCACAAAAATAATTTATAATATTAGTTTAATATTTACATATTTC
CA1032(SEQ?ID?NO：92)
NGGCGACGGCGATTGATTCTCGCCCTGTGGTGGAATTCCAGTTTGACCAGGACTGTCGTAAGCTTAGTTTAATTTCACCAGCAGACACCGACTCAATAGCACCCTGGAAAAAACACACTCAAACCAGAAGCAAATGAACTATAATAGTCCAAGTAGAAGAAACACAATCAATCATCCAAGAAAAGATACTACTACATCACCAACAATACTGCTAGATAATGTAAAAAATGGACAGAAGAAATAAAACTACACTGGTCTTCCACCGAAAGAGTCCAAATAGAAACACAAGGAATAAAGCAAAAGAAAACTAAAATTACCATAGCACTAGCAAGATAATGTAAAAACCTACATTGATCTTCTACAGAAGCAGTTTGTTTTATTTTTTCTCCGTTTAGAGAATTTTGGGGTGCTTCTCACCTTA TTGAACTTG ACGACGACATCCCTGAGGCATTTCCAACCGCCAAAACGGAACACAACAGATGCTCCCAGCACTCGGCTAAGAATCCATGCAAAGAATCTTGAACAGAGTCTGGAGAGTCAAAATG[AAATAAATAAATAAATATAATAATAATAATAATAAT]AAGACCACTATAGCAGCATAG TCCAGCAGCTAAATCATGC AATCTCAGCTACTGAAGGAAATTAGAGAATGTGCAAACCGAACTANAATCATCACTAGAACTAACTCACACGAAGATCATCCACAAGACCATGGAAAGAATCAGGAAC
CA1034(SEQ?ID?NO：93)
AACGGAGACTGATATCTCGCCCTGTGGTGGAATTCTCTGACAATAGTAGAAACTAA AAAATGCAACCATGCAATACGTGGTTTTATAATCATTCTATTGTTAAATATGATGAT[AATAATAATAATGATAATAATAATAATAATAATAATAATAATAATAATAATAATAAT]ATTACAGAATGTTGATGTAATAAACAAAAATAGTTTGTTAGCTAACGCCTCAGATCGATCAATGAGTAATTCATTCAGTTACAACATAAAGAAACAAATAAAAACTATGATAAAAAAAGTTTTGACATCATTTTTTATTGACATGTCAATATGTGATAATACACTCTCT G?CAGCAGTGACAAACAAATACTACAAACTCTTATTTTTAATCGTTCAAAGATAAGAGTCTATACTAGTAGACTAGAAAGTGGGGGGAAACAAATAAATTTAGGAGGATTCATTGACAATTTAAGAAGACATTTTTGATACGCCTCGTCTTATTAGAATTGGGAATGGCCTATGGAGAGGATATGAATGTGATGGGCATAGTGATAAGGTAGAGGAGATAATGCAGAAAAGCGAGAAGAAGAATCTTAAACTATCATTTATGAATTATGAGTTAACCTCAGAAAGCCAGTTTACAAAAAAAAAAAATTATGATATCTCCACTCGTTTCTATTAACTTATTCCTCCATGATTGGTCGTTTTTGTAAACTTCTGATGATTC
CA1035(SEQ?ID?NO：94)
AGCGGAGATTGATTCTCGCCACTGTGGTGGAATTCAGTAGCTATGTGCTATTAGTGCATTGATTGTTCTTTTGTGTGGTGTAATAGACACCTGTTTGTTCCATGCTAGAGCTAGGCCTAAATTTTTGTAGTGCTATTAACTAAGTCAGTGGTTTGTGGTTTAGCATCCCATACCTCACTGAGTGACTCCCTTATTGCTCACCCCTCCTTCGTT CTCCCAGGTGAGACCGACAATCATGAGTGATTTTATCGGATTGGTACTTTTGAGCTTTTATCGTTACTGAGCTTTTAGACCTTTGGACTTTTATCTTTTATGCTATTTCATATTTCAGACTTTCGGTTTTATATTGCTATCTATATTTCAGATGTTATCGGACCTTCTGATATTGACTTTTGTATTATGAAGTGGAG[ATTATTATTATTATTATTATTATTATTATTATTATT]AC TAGATTCCTTTTCCGCGCTACGCGCGGATAGTATCTTATAAATTTTAAATTTATTTTTAAAAAAAAAAATATTTAAAGTTTAATTTACATTATTTTATACCAAAAATCACAAATATGGCTAAGAATTGGTTGATTTTATTTGTGATTTTTTGACAATATTAAATTGATTTATTTATTGCTCATAGTTAACAGATTTGTTTGATTGGTTTTCAGTTCATAGTAATGTATAGTATTATATTTGGTGAGTGTATATA
CA1066(SEQ?ID?NO：95)
TCTCAGCGCTGAGCTCCATGTGGTGGAATTCATCCTAAATCTCTTTGATGCGATTGATCTCTTTTATAACTCATTTTGACTCTTTTGGAATTGGAAGAGATGGACTGGACGGCTATTGCAAAGCCTTTGGAAGAGATGCAAAGGAGCTGGCTATTGCAATCTTCTACGCTCAAGAATAAGAAGACATATGTCAAGAAGAAGAAGATATATGCCTGGA CTCTTGCCTTCATCGGCGTACTTGTGGTTATTGCATTTAGTTTGAACATAAAGCTCTTAGGGGCTCATGCATAACGCTTTCTTAATTAGCTCTGTTTTTTCCAACTGATGTTTACTCTTTCTGAT[ATTATTATTATTATTATTATT]AATTGTTAGTTGCTGTTGACCGGTTG AGGCTGTTTTTGAGCCCTGGATATCTTTCTGAGTTGAGAAAAATTTCATAACCAAATCGAGATTGTTATGTGCTCTTTCTTGCCTCCTTTCAACAAGTTTAATAGAACCAAAGGCAAATAGTTTGTCTTTATTCTATACTAGGATTGCGAATCCCCGCGGTCCGCGGGGAAAAAAAGATGTTTTACCGCAAAAAAAATGATGTTAAAACTTAAATGTAATAGTAAAATTTTAGTTTGTAACAATCAACCGGTTGAATGGTTAATAATCAAATATTGCAATGTTAACTATTAAAAATTACAGTGGAACATTTAAAAGTTGTGAATATTTATATAAAA
CA1080(SEQ?ID?NO：96)
AGCGGCAATTGTATCTCGCCCTGTGGTGGAATTCTGTGATGATCAAAAACAAGTTTCAATCCAAATATCCTGATTTTGCAAATTATT TGACAAA ATCCACATCTCTAATGGTGTTAGAACACTAATAAG[ATTATTATTATTATTATTATTATTATTATTATTATTATTATTATT]AACAGTCTTTCCAAAATAAATGATTGATAAAATATTGAAAAAGAAGGCAAAGAAGAGGTTGAGAAACAATTCTTATTTCAAAATTTTACAAAAATACAAATTGTTCGCGTAACATTTTCATTTTCTATTTAGTTTAATTTTTGTCATTTAAAATTATCTTGTTGGTGTTGTTGGCGT AAGCCCAAAACCGATGTAGCCTACACTGGGCCAATCTCCTGCGCAAGCCCAAGACATAAAGCATTAGGGTTTTGTTGCTAGCTCATATGTAAACAAAACTTAAGCTATCTTGTTGCCTAAGGTTTTAAGTTTTCTAAGATACAAAGCTTGTACATACACAAGCTAGATCATAGTTGTGATCACCTCTGTACTCTCTTATTCATAGTGAAGTTTGGGAGGACAGTCTCCCACGAGACGTACCGGTTAGAGGCCGGGAACTCGTTAAATTGTGTGTGTTCTTATTGCTTTAGTTTAATCTCTTCTTAAACAACCATAAGCATGATAAGAACTAGTTA
CA1090(SEQ?ID?NO：97)
GGAAACGGCAGTCTGATATCATCGCCACTGTGGTGGAATTCTAACATAACATATGATTGATAGTACATTGTATATTCTAATTCAAATTAAAAAATATAAATATACAAATATAAATCACCAATATAGTATTTTTACATATTAATTTTATAATGCTTTATTCTAATATTTTCTTATACCTACTTATTAATTTTTAACTTATTAATTCTAATCAATGGTCTATTCTGTATTTATTCTCATAAAGAGAAAATAAAGATCTACCAGAAATTGATATTTATGTACATTCATTACATGTACAGTAATAAGACCCACATAATCATTTTGTTTTAGCTATGGCTCGTGTTAGGAAAAATCTATAAGATGTATCTAATAGTGTGTTGCAATACTAGACTACCAGTTGATCCAGTTGTTTCAAATAATTAGTTATGTTTCGGAAACTTTGTAGATTGGCTTATTTT CCATGCAGCTTTTTGTTACGACAGAACAAATTACGCATAAACCTTTAGGCTGAGCAAAGTTGATGACTTTAACCAAGATTGAGTCTTGAATATGTGTCATTACATCGAAATATCGAATGTTAAAAATA[TAATAATAATAATAATAATAATAATAATAA]CAATAACGGGTTCATATATCATTACAATTATAACGTAATAGCTGAAAATTCAAAATTGACTAAAATAATATTATGACCCGTCCCTTTTTATGGTTCCCCCGT TCGTGTATTTGCATT GTTGGCCGTTGGTGATCCCATGNCGCACTTACCCTCCAAGTCTTCA
CA1097(SEQ?ID?NO：98)
TCTGGAGCTGAAGTCCATGTGGTGGAATTCTGATTTTTGAAAAAATTAAGCGTTATTTTTGTGATTTTTGACTTTGAGTGCTAATTTGGAAACAAAAACTTGATTTAGAGATATTTTTGTCTTTTTTTCTTCTCCAAGCGTGTATCTTTATTTTTATTTTTTATAATTAAACAGGCGGCTTTTTATCCTAATTCAATTCAGGTGGGGTTTTGTTCTTTTACACATGTCAATTTTGTTCTTTTAATGGTAAAAATTTAAGATAAATTATAAACTGAACCGGAATCGCAATTGGTAATAAACTGAACAAAATTCCTAATAAGATATATTCCTGAAAAAATCCCCGGAAGATTTTGATACGTATTAAAATCATATTAAGTTTGAAATATCAAGTTTTATATAATAAGATATACATTATATGCAACATCTTTGAATAACTCTCAACCTTTTGGTCATATCACAAT AAAGTGGTGGAGCTTTTTCCAGTTACTGATGAATGAGTTAAAAAGTACTTAAGTTGCAATAATCTATTCATATTCCATGATCAAAAGCTCTTACAAGAAACAAAAGATTACATGAAAATGTCCAAAAGGGTACTTT[ATTATTATTATTATTATTATTATTATT]ATCAGGATTGAGACCCACGTATACGTAATAAGAAAATATATAACTAATAGCGAATGCTACCTTATGTCATATACACGTA AACACATCCCACTGGTCTTGGCAACACAAGGTGTCATCCTTCTCTTAAACATTCAAC
CA1107(SEQ?ID?NO：99)
AGACGGAAATCTGATATCATCGCCACTGTGGTGGAATACTAAACAAGGCATATAGCATAATATTATTTCATGATATAAAAGCAAAAAAAAAATAGAATAATTAAATATACAATAAAAAAAATAAACAATAACTAAATATACAATAGCAAAAATGAAAAAAACTAAATGAAACATCTATCTGAAAAATGTATAATAAATAAATAATAAGTAAATATATAATATGAGAAATAAAAATATTACACTAAATATCTATCGTAATATTAAAATAAAAATGGAGGTGGAGGCGTTAATATGGGCAATGGAGTGTATGAGGAATTTGCGTCAGTTTCATGTCACGTTTGCAACAGATTTTCCTCAATTGGTGAAGATGGTTTGAGAACCAGAAAAATGACCAGCATTTGAAAGTTATCTAGAAGACATCAAGATTTTGAAAGAAAGTTTCATCAACTCAGAGATCATTCATGTACCTCGGACGGAGAATTTAAGAGCGGAGAGTCTAGCACG TAGTGTCAAAAAACATTTGTCT TTCATCGTTCACATGGATTTAGAGTTACCAGTTAGGTTTACAAAGTCGGTATGAGTCTGTAAAAGTCGATTACAA[AATAATAATAATAATAATAATAATAATAATAAT]AAGTAAATATATGATACAAAAAATAGAAAAACTACATTGAAAATGTGTAGTAAAATAAATAATAAATAAATAT AAAATATGATGA CAAAAAAATGACAAAAAAGTAAATATAAAATATAACAT
PE0012(SEQ?ID?NO：100)
ATACGCCAAGCTTCAATCAAAGGAGGGAAGTGGTGAGAATACAAACCTAGCCTTATTCTCTCTATGTATCTTCCTCAACTCTGCATCTTCCAACTGTGCCTGAAAAAAGAAAAGCAAAACCCATTAGACGCTAAGCTAATGCAATTTCGAGTTTAATGTTTCAGCTTAATCCACATAAAGACGGAAACATACCTGCTCCTGGGTGAATTCCTCCGGTG CACCGTCGGAGTCTGAATCGGAGTTGTGATCTTTGTTATCCGACATCTGATTATTAT[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]CGTTGCTTTCTTCTCCCTCAGACGACA CACACTA NGGTTTAACGGCTCTTCAGTTTCTCAAAAACAGAAGATTTCTATTCTGAGAGTTAATTGCTTCTCTCCTTTATGGTGGATTCTATTGGGAAGCTTGCATGCCTGCAGGTCGACTCTAGANGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCTGGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCTTTCGCCAGCTGGGGTTANTANCGAAAAGGCCGCACCGATCGCCTTCCAACANTTGCGCANCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCTTACCTCTGTGCGGTATTTCCNCCGCNTATGGTGCCTCTCANTACAATCTGCCTGATGNCGCNTANTTAACCANCCCGAAANCCGCCANNNCCGCTGAANCCCCTGAAGGGGNTGTTCGGCNCCGGNATCCGCTTTAAAAAAANCTGTTAACGTCTCCGGAACCGCTTTTTTCAAGGTTTTCCCGTCTCNCNAAN
PE0017(SEQ?ID?NO：101)
CGCCNNGCTTCCTTTAAGAGTTAATTTCATACAAATAAGCCCACTAAACAGCGTTGTTTAAACTTTAAACCCTGTCTTCTTACCAAAGCTCCTCTTCTTACATAGTAGCTCTGCTAAACCTTCGCCGCAATAAACCCAAAGTCGTAAGAAACCGTCGCCATGCCTGCTCTTACGCGTAACAAGCAGAAGGGAGCTAAGTCGCAGACTCCTCCACTGATTAAGCGGACTAAATCGAATCCCACGCCTCCACCGAAGAAGGCGATGAAGTCCCGTAAGCCTCCGTTGAAGAAACAGAGGAAAGGTGTTTCGGATGAGAAGCCTGAAGTTTCTAATGATGAGGAGGAAGAGGAAGAAGAAGAAGAAGAAGAAGTGAGTGAAGAGTCTGATGACGGGAGATGA ATTGGGTTCTGACCTTTTCTCAGATGGTGACG[AAGAAGAAGAAGAAGAAGAAGAAGAAG]ATGATATAGAGCCTTCGGATGACGACTTTCTTG GTGGTAGCGATGAGGAAAAGGGAACTTTGGGTTCTGATTCTGACTCTGATGAGTCAGATAAGCTTGCATGCCTGCAGGTCGACTCTAGANGATCCCGGGTACCGAGCTCGAATTCCTGGNCGTCGTTTTACNACGTCGTGACTGGGAAAACCTGGCGTTACCACTTAATCGCCTTGCAGCACATCCCCTTTCGCCNGCTGGCGTTNTACCNAAAAGGCCGCNCCGATCGCCTTCCACAGTTGCGCNCCTGAATGGCGAATGGGGCCTGGATGCGGTTTTTTCCCCTTCCCCTCTGTTGCGGTTTTCCNCCGCCTNTGGTGCCTCTCNTTCATCTGCNCTGATGCCCCTTNTTTANCCNNCCCGAACCCGCCANNCCCGTGAACCC
PE0063(SEQ?ID?NO：102)
ATCTCTATCGCTATACTTGTCACTTTGTTCACTTTCTCAGCAGTTCCTTCCACATGGTGATGCAACCATCTCGCCATTACACTCAGCAAGCCTCCCTTAGCCTTCTTGCA TCGTTCTGACCTGTCGTTATATCCATAATTCTTTGCAATTTTTGTCATTCT[CTTCTTCTTCTTCTTCTTCCTCTTCCTCTTCCTCTTCCTCTTCCTCTTCTTCTT]TATG AACATGAGCAGCCATTTCCTGTCAACTCTTTGACGGATGTGCACCAATTGACAAGAGCTTACTCTGTTACACCACTCCAACAAACTCTTCCTTCGTCTCTTAGATATTTTCTCCATTTGTATTCCCAGTGTCTCAAAGTTATCATTATCCTTTTCGCTCAAAGTATCATCCAAATGCTTTTCAATATCTAGAAAAGTTTTTTTCCATCTCTTTCTCTTCCTTGTTACTCTTACACCTCCCTCAACCATTTGATCGAACACATGGTGGGCATACTTCTCCTTCTTGGTCTCAAGCAACTCCTCTCTTGCATCNTTACCCCTCTCAAAGT
PE0091(SEQ?ID?NO：103)
CGGCAATAATGGACCACTGGTTTGGTTGAACCACTAAAAAGAGTGC[GTGTGTGTGTGTGTGTGTGTGTGTGT]GAGGGACTCTATTTAAAAGCACTGCTTAACTCAATAATTATTCCATCGCTCCAAAATAAAANAGAATAGCTAAAAGATGGCTCT CGAAGTCTGCGTGAAAGCCGCCGTTGGTGCCCCTGATGCTCTCGGCGACTGTAACTTCCCTTCTCTCTCTCTAGCTCTTTTTTTTTATATCAGATTATGATCTCTGATGATCTTCAAATGTAAAATTTATAATACATGATTTGTCTGTCGTTTCAGG
PE0131(SEQ?ID?NO：104)
CCGATAAGAGCCATCATCTCGAGGAGGGGTATTAAGGGATATGTATCCCACATTGGAAAATCAATGGGACATTAAGTAATATATAAAGGGTTAGGGCCAATCCACTAATAGCCAATTGGTTTTGAGTTGGAAGCCCATAATAAACCCGAATCTAACAAGATTTTAGATTGATTAAGGAAATTAATATATTATATGCAATATTTCATGGTTAACGTCAAAATAAGTCCAATTTATAAACAAGCGGATAAACATTTCCCTATATA TATGGGAAGGTTTGTGGTTGCCAAACTCAAAGCACATTGGGTCTTATCTCTCTCTAA[CACACACACACACACACACA]AACTCACGTATATATTTAGAGCTA[GAGAGAGAGA]TGGGTGAAGAGATGAAAGA AGTGAGAGTAATCGAGGAGTGGTCTCCGGTTATAGTAATGGTGAT
PE0133(SEQ?ID?NO：105)
CC TCCTGTGCCAAGTTTTACAAGGCACCCAACCTGTGAACCTAAATTGCTTTGAAAGAGAAGTTTCTCTATCTATAT[CACACACACACACACACACACA]AATCTAATCTTTCTCTTTCAACCGTAAATTTTGCTCACCACCAAGGCAAGTTTCCTTCTTTTGCTCCCCTAGCAATATTAATTGCTACTAA A ATATCTTGCTAAGGGTAACCAAATCTTGCTTCATTCCTCTGTAATATCACAGAAAGAAACTAAAATTTAGGGTTTTTTTTTGGGTTCTTTCCATGTGATGTGAGCATTTTTGGGTGAGAAAGATGAAGACTATAATTAAGTTAGGGATTGGGTTGAGTTTGGTGTTTGGGTTTCTTCTCTTAGCACTTATTGCAGAAGTCTATTACCTTCTGAGATGGAAGAAGCACAAGAAGAGAGTCATAAGCCAAGAGAGTGAGGAAGAGAAAGAAGAAGAGCAACAACAACAAACTGGGT
PE0177(SEQ?ID?NO：106)
TACCCAAGCTTCCACGATCGGAGGGATCTATCGGGACTTCTCGGAGATTTGTTTCACGGTGATG TCTATTGATCTTTGGCTCTCTGTTTCGATTGTTGTTGTC[TTCTTCTTCTTCTTTTTCTTCTTCTTC]TCGAGAGGTTGCGGGGTTTTGAAATCGTCTCCTTCGTCCATGTAGTCGTTTTGGTGTTTGTCCGCCAT[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GAGA] GCGAAGACGTTACGAAAAACTTCGATAGAGAGTATAAGAGAGAGATGCTGAAACTGCTTAAACCCTAATTTTGATCGAGTGTGTTTTGGGAAATTTGCAGAGTAAGTCCTTATATTTGAGCCGAATTAATTAAAGTAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTTATAGCGAANAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCNCCTGAATGGCGAATGGCCCTGATGCNGTATTTCTCCTTACNCNTCTGTGCGGTATTCCCNCCGCATATGGTGCCTCTCNTTACATCTGCTCTGAAGCCGCNTNTTTANCCAGCCCGACACCCGCCAACACCCGCTGACCCCCCTGAAGGGCTTGTCTGCCCCCGGGNTCCCTTNCAAACAACTGTTNACCGNCCCCGGGAACCGCNTNTTTCAAANGTTTCCCCCGCCTCCCCGAAACCCCCAAAAAAAGGGGCNCTNANACCCCNNTTTTNNGGGTTANGTCNGAAAANAANGGTTCCTAAANTTCGGGGGGCCTTN
PE0187(SEQ?ID?NO：107)
ATCTAG GACCCCATGATCCGAATAAGGATAATAAAAAAATGGATCTGCCGAATAAGTATCTGGATAACTTAAAATTCCCTAGATACCCCCCCCCCGC[CACACACACACACACACACACACA]TCAATTTTCCTTGTAATTTTTCTCTTGTTCTCTATTTTTCTA AACTCCAATAAAGCAAGTCTTTAACATATACTCCACCTTTATTTGAGTAAATAATCATGGATTTAATCTCTAAAGTGAAGGACACTTTGTTTATGTTTTCTGTTTTTCTAAATTGTAAAATCTATTTTCTACCTTTTTAATGTGCTAATTTTAGGAAAAATTATATCAATATTTTGTGTCGTAATTAAATCTGTCAACATGAAGTAAATCTGTGTCAAAAAGAAAAAAAAATCTATAGAAACATAATTAAAGTAAATGTATGAACATATAAAATAAATCTATGAATGATGTATAAATCTATCAAAATTAAATAAATATGTGG
PE0203(SEQ?ID?NO：108)
CTTTAATGCTTAAAGCTCGTTCCAGCTCAGGGGTTGGGAGTTTAAGCCTAAAACTCGAGATCTGCTCATCCGCAGTTGCTGATTTGGCAGCTTATTCACCTTCGCATACCTACAAATCAACGAA TACAACGCAAACGTTCCTCCTG[CACACACACACACACACACACA]TATACTTAGAACCACATAACACCACTTTTTACAAAAAAAAACAAGGATTAGGGATGTAATATTATTACCTTCGCCATTGTCGTTGGCTTTAAGGACAACAAAGACATACTTTGCTAAAGGAATGACAGCAATGGTGTAGATAACGAGAGACA AGGCACCAAGAACA TCAACTTCTGATCTGATAGGAACTTTACTGAAGACATCACTAAACACATACAAAGGGCTTGTTCCCATGTCTCCATACACAACACCTAACGTCTGAAACGCTATCCCAATCGT
PE0250(SEQ?ID?NO：109)
ACTCAATAACATCAGGCTTTTCTGGATGCAA?TCTTGCATTGGTTTGGTCCAGTTTGCATCGGGACAAATATTGTTGC TGGTGTATATGGGATCG GAATCTTTGCTCCGACATTCTCTTCGGTGTCTT[CATCATCATCATCATCATCATCATCATCATCATCATCAT]CAGTATCAGTATCATCTCTTATTCCAAACTTCTTATATTGTAGATGTTCTTGACCGGAGTGGCAAATGAAGACATCTCATATATGGCATTGCTTTTAGGGTTTTTAAGAAGATGTGGTGTTGCTATGTTGGTCTTTGATGCAACCACGGTGGTCGGAA?TGGG CGAGAATGGT CTGCAAACTAATGGAGATTGTAGGTGTTCACATGGGTTCATCGACTATTACTTTGGGAGAAGTCCAACAACATCAAGCCCTCTTCTTCCTGCAGGAAAATAAAATATTTCGTTCATCAATTAAAGTAAGAGAATTAACTCATCACTATGGTGATATGTTAGTTTCTGTTTATTGTAAGACTTAAAATTACCAG
PE0281(SEQ?ID?NO：110)
ACAAAACCGCTTCCACCTGCGTTTCGTCGCAGNCAAGCATTTGCGTTTCATTGGGTTTTCTCGGTGAACCAAGCTCGCGTTAGTATCCAAACATGTTTTCACAGAATCTCGTAGTAACGACATTTCTTGTTGAAGTTGTTGGATCTGTGTTCTCATTTCGCTTATCAGCTCCATTTCCTGAAACGTTTTTAATTAAGCGGTTCAATGATTCTCTTCTATGGGGATAACAATAAAATCTAAAAACCTTACAGGTGACGGAGGGTTGTGAACAGACAAGACAGGAGTTGAAGTTACTTCAGTGTCTTGACAACTCCATGATCCTGCAG GAGACGATGCA AAGATCGGCGAAGAAGACGAGTCATCTCCATCGTCTTGCTCTTCACCTTCTTCAGTAAATGGCT[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]CAGTTCCTCCACATTTTCATTCTATGGTCTTCCTCTTCTTCTTCATGTTGCAATTCCCATTTTTCAGAATGTT TGTTCGAATGTGTCTGCACACGAGACATCATGAGCCTATCGATCTGATCTCTTAAACCGGTCTGGGAGAANGTCTGTGACCGGTCCTCTGTAATATCCAAAAACCACACATTTTTCTTAGTNAATGGGTACCCAATTAGGAAAATGGGATTTAAAAGATTGGATCAG
PE0283(SEQ?ID?NO：111)
AATGAACGAGACAGGAGGTGCCACCCTATTGTCTTTGATGGACCAGAGTCTAGCTATCCGGTAAACAGTATTTTATAACAAAGACATGATCATGAAATGATTTTTTTCTTCTGAAGTTTAACTGATGACTCATATATCTATATCTGACTAGTTCATCGTGGACATGGAG CATTCACAGGACCAGAGCACACATCGAAGCTGACATACAAATGAATCTCTTATTGCCTCTAGTTTCACACGTAAATATTCAGTTTCTAG[GATGATGATGATGAATTGATGATGATGATGACGATGATGAT]CAGTCAAAAGTACTGTAAATTG ATGGTTATGGTTGTC TTGGCTTTGCTTAATCATGT
PE0286(SEQ?ID?NO：112)
ACAAGTTACCATTTGAGGATATATCAGAGAAGAAGGAGTTGCTTGAAGATGACGAGAAAACCAAAAAGAAGATGAGTTCTAATGGTCGTTGGTACGAGGAGCTTGATGTCTTCATAGAGAAACCTGAAACTGGTGTTCTTACTGGTGATGGTGCTGTGGTGGACGCATGACTGGGAACGAACCTGTTG A TGGTGACGAGTTGGATGTTGAGCAACAAGATGATAATTCTGATGGTGATCATGGTGATCATGAAGCAG[GAGAGAGTGAAGA]TGAGTATCAAGCGAGTGATGAATCTGATAAAGAAGAGGATATTGACAGAAATTTTGAAGAGGATGTTGAGATGTTCCAGGGATGAGAACTACGATGG AGGAGATTCCAG ACGAGGAGGAGGTATATTCTGACACGGAGGAGTCATCTGATGATGAAGAGGAACAAGCTGAGAAGGATGCTAATAGGGGTGAATTAGATGGCATTTTTAAGTCTTAGGCAGGAANTTGCAATGCCTGCAAGTCGACCTCTAGAGGATNCCCGGGTACCGAGCTCGAATTTCCACTGGGCCGTCCGTTTTACAACGTCCGNGACTGGGAAAAACCCTGGGGTTAACCCAACTTAATCGCCTTTCAGCACATNCCCCNTTCGCCANGNTNGGGGTAATAGCCNANAAGGCCCGCAACCGATNGGNCCTTTCCCAANAGTNGCCGCACCTNAAATGGNGNATTGGCGCCTTANGNGGGAANTTTNNCCTTANGNATT
PE0324(SEQ?ID?NO：113)
ACATAAGCCCTTTTTATTATCTCTGCATATCATTACATTCATTTTATGTCACATATGTTTATTGCTCTTCTCTTCAGATTACTATTACATCGCAAGTAAAACAAAAGAGTTAGAAAATAAAGTAAACACTCCATACATAGTCAAAGTATCTCCATTACTCCTCTTCTTCGTGTTAACAAGTCTTTAGGCGTTTCTAAACCG CAGAAACCATCATAGCCGGTGATGCACCAACCATCAAGT[CTTCTTCTTCTTCATCATCATCATCATCATCAT]CCTCTGCTTCCCACATGAAATGAGCGTATGATCCCAAAACCATACTACAAAAGTCACAAACCTTTAACATTCTGAAAAAAAAACTCATCAAAGAATCCAAACTCTACATATAACATAACATACCAATCATCAGGAGAAGCGTTAACAGCTTGATCAAAGTAACACTGAGCTCTCTTCTCATCTC TCTTCGTCTCCCAAATCAGCTTCCCATACATCGACAACGCTTCACCATCACCTGGATCCGCAAGTATAGCTCTCCCGTAATACTCCTCCG
PE0340(SEQ?ID?NO：114)
CTTAGTGACCCAAAAGCCATTGGTGTATGATAGAAAAGTTAGTTAAATAC CGTTACTCGCAAGGAAGACCACACATTTTTTAATTCTATCTCACTTAGTCAGACCAGCTCGGATCCTTCTCTAGAAC[CACACACACACACACACA]CTCAGAGTGAGAGATTCATCAATGGCGGTTTCTTGCAGCCACTCATCGATTCTCTTGCCCCCAACCACCTCCTCCGTTGGCTTCAACCGCTTCCCTTGTCTCCAAACGCTGCGTTTCAAATCCAGAAACGTTTATCAGAAAGCGAGGATCTCTACAGTGTCGGCGTCA TCTTCACGGTCTCTCGAAGCTCTGATCTTCGACTGCNACGGTGTGATACTCGAATCGGAGAATCTACACCGTC
PE0355(SEQ?ID?NO：115)
TCAAAGAGCACTTACAAGGATCCAGACG ATGGAAGGCAACGATT CTTACTCGAACTTGAGTTCATTCAGTGTCTCGCGAATCCTACTTACATACACTGTAAGCTCTTATGATTCCTTATCACATAGTATCTACTTATAGCATTTAGGAAGTGATAAGAGATCTT[GTGTGTGTGTGTGTGTGTGTGT]TTTATGCTCTATGATGAACTTACCACTTAGCTTTTNGATTCTGTTTT GGCAGAC CTAGCACAGAATCGTTATTTTGAAGATGAAGCATTTATTGAATACTTGAAGTATCTTCAGTATTGGCAGCGACCAGAGT
UB0015(SEQ?ID?NO：116)
ACTTCAGTGGTCGAAAATCAAAATATTCTTCCATCATTTTAGTTTTTTTTTTTCTCTATGTTCGCATCAAGAAAACGAAATGAAAGGGATTATAAAAGGAAGAAGAACTTGTGAATCACGGTAAGTT TCGGGGTTTGTTGTGA GGAGATTTCGAGAGAATCAAGAATAAAATTATATCACGAGATTTTTTTGTTTGAAGTGAGAAAGAAATCAAAGATTTTATTTTTTCTCTTTTGGTGAGTGATA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]CGTGTTTTGGAACTACGGTGATTTTTACTATTTTGATGATGTTTTCAACTTTGAAGAAGACCTTCTCTCAT GCTCACTCTTAGCATCCTCCTCATTTATAGGATTAGATGGGAGAGAGAGAGCGTTTTAGCCATTAATACTTTAATAACAAAATGAAAAATCTGATATTAACATTTCTTTTTTCACTTCTCCATCAGTGGCATTTTCGATATTTT
UB0126(SEQ?ID?NO：117)
ACCTCCTG ATGACTGCTTAAACAGCGCCTGCGAAGATCTGGATTCTGTAGTTAACCAGGCTAGGGAGTTCTTAGAGGACTGGTCCCCAAAGTTGAGCAAGCTCTTTGGTGTAAGTTGATGAACAAGCTCTCATTTTCAGTTTTCTTT[CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT]TTGCATCATTCATTGAGTTGTGTGTGTGCAGGTGTTTCACT CCGAGCTTTTGTTGGAGAAGGTCCAGACTTGTTCACTGGAGATTAATCGCATACTTCTTCAGTTATCACAGTCAAGTCCTGTAACTTCAAGTGT
UB0163(SEQ?ID?NO：118)
ACAGAAACAGTAACATCAACACACACAACAAACAGCTCGCGAAATGAATTACAGATTCCTCTCCGAAATCAAAACAGG?AAACGGACACA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGATGAGA]AGGTGATACCGTCGAGAGGTTTGATGTTGCCGTCGCGGCGATCGACGGAGAAGCCTTCATGAGGCGAATCGACGGGTTTGACGACGT
UB0181(SEQ?ID?NO：119)
ACAGACGCGATGATGACACTTCTGGTCCTAAGCGTTGTGTTACGTTTGATACTCCTGCGCTTGTTTACTTGGAGTATTCTGATGTGGTTGCTGATAAGTATGAGAATCTGAGTTTGGACAGCTTGGTTGAAGTTAGGCTTGATCTTCAGTTGACTGCAGATCAAATCATGCGCAAGAATGCTACAGACAGTGTTGGTTTTGTTCCCGGTGATGTTTCAACTTTGTTCATGGGGGTC AAGA ACGTCAAGATCCTCTGCTTATCTCCTGATTCTTTAGATGTGAGTCCAGTCCTTTTTAAGTTAGCTTCATCACTGTGTAGCATTTGTTTTTTTTTTAAATTTGATTGGTTAGTGATGATACAAAATATTTGATTCTG[GTGTGTGTGTGTGTGT]TTGTGAAAGTTCAGTCCCTTTAACTTAGCTTCATGAGTGTGTAGCCTTTGTTTTTTAATTGGTTACTGATGATATGGTGTGTGTGTGTGTGTGTGTGTTTCAGA CGCTCTACTACCGTGGTGGTGACATGCCGGTGTTCAACAATCTGATTT
UB0196(SEQ?ID?NO：120)
A CATGAGAACAAGATGGGTTCGAATTACCTCTAGCCTAGATCTGGATCTGGAAACAAGAGACAAGGGAGAGACGAGATCTTGTCACCACCACCAATCGGCTGCCACCACCACCACCACAACACGGCGCCAGCGAAAGGGAGATA[GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA]AGAAGAAGAAGAGAGAAAAGGAAAAGAGAAGCTTGATGGCTAGGGTTTCTTAGTCTCTCTAAATCTCTGCA GGGCTTTGCTCAAGTTTCAGAATGAGAGAAAAAAGAGAGGAGGCAACTTTATTTATAGGAAATGGAGGGAACCCTAGGTCATTTACCTTAATGGGCTGCAGTCCTAACGAGCTCTCGTTAAAAAAATTTGGGCCGGGTATCGGGATGTTACACTAACGGTGTGTGGCGATGAAGGCTCTTCGACTCTCAAAATTAATGATGTCCATAACTAAATAAAAACTACTCGACTTTATTAAGATATAGCTTCAATGATTTAAAATTAAATATAGAACTCT
UB0307(SEQ?ID?NO：121)
ACCTN TGGGTAAGTAACTGTGGTGGC[CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT]CAATACACTCTTCACTTAATAAATGTGAANACGTTAACTNGTTTCTTTTNTCACTTCTCAGTTATGTA GCTCCAGAGTATGCGA ACTCTGATCTTCTGAATGAGAAAAGTGATGTCTATAGCTTTGGTGTTGT
UB0315(SEQ?ID?NO：122)
CACGAAAGCAGGCCCCACCCAATAAGCGATGAGCTGTATATTTATTTTGTCTTGTTTTCACAAAAAATAACCCTTCATGTTTACAGTTAATTACACAACAGCCCCTTTCTTTCC TCCATGACCAACGACAAGGTCGAATTT[CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT]CCGTCGTCTTCATTCAATCTATCTCAGTGATTTACT CGCAATAGAAGTCGCCTCTTAATCTCTCGAGAGAGAAGCTCAAGT
UB0331(SEQ?ID?NO：123)
CGTGGACTAACGCTC GTGTGCAGGAAACGATGTTCGTGAAAAGGTATCCGATCAGAGGAGCCTCCGCCGGTAAAAACCCTTCG[CCGCCGCCGCCTCCG]TTGAATGGTAATAACTCTTGTTCT CGCTTTCTCTTCAAACTCC CTTTTTTTTCTCTGATTATTTTTGTTGGTTA
KK66(SEQ?ID?NO：124)
GAATGGGAAGCATCACAATGATAATGCTAATGGCGGTTTTGGTCTGGTCCATTACTCTAGAGACCTGCATTGCTAGAAGAGGAAGACATTGGAGACATAACCACCGAAGCTCCTC[A/T]GACTTGTCTGATTCCTTGTCAAGCAAGAAACCAAAAAGCCACAGTCACCACCACAGCTCTCA[C/T]AACAACAACCATAATCATCACCACAAGTCTAAACCTAAACCAAA[A/G]CCAAAGCTGAAAACGCCGCCAAAAAGTGACCACA[A/C]TAAATCTCCGGTGGTTTCACCGCCACCAAAAGTCCAACCACCGTCTCTTCCGCCGCCAAAGGGATCCAAAGTTTTCAATGTGATGGATTTTGGCGCAAAGGGTGATGGCAAATGTGATGACACTAAGTCGTTTGAAGCGGCTTGGGCAGCAGCTTGCAAAGTGGAGGCATCCATGATGATCATACCGCCTGAATACACTTTCCTTGTGGGTCCAATCTCATTCTCTGGTCCTTATTGTCAAGCTAACATTGTGTTTCAGCTTGATGGTACTATTATAGCTCCAACGGATTCAAAATCATGGGGAAAAGGGTTAATGTGGTGGCTTGAATTCACAAAGCTGAAAGGAATTAAAGTACAAGGTAAAGGTGTGATTGATGGAAGAGGCTCTGGT
KK98G(SEQ?ID?NO：125)
GACAGAGATAGCCCTAACTTAGTCACTCTCTCTCACACACACTCCAGTTCAAAGTTCAAA[A/C]AATGGCTCCTCCACAGAAGCTCTTTCTCGCCGCCATTGTCGCTGCCGTCATTGTAGCCGCCACCACCGGATATGCACCTAATAGTGCTGCGGAAGATATTGTGCATTCCTCATGCGTGCACGCGAGCTATCCATCGCTATGCGTCCGTACACTCTCTACCTACTC[C/T]GGTCCAACCATCACAAACCGTCGCGAGCTAGCTCAAGCCGCCGTCAAGATAAGCCTCTCCCACGCTCGAGCAGC[C/T]GCTAAGAAACTCGCGGCTGTGAGAGAAACCGTGGG[A/G]AAGAAACGGGTGAAAGCGGCGGTTGTGGACTGCGTGGAGATGATTGGAGACTCGGTGGACGAGCTG[A/C]GCCGCACGCTAGGCGTTTTAAAGCATCT[A/C]CACGTTTCGGGCGTTTCGGCGAACGAGTTCA[A/G]GTGGCAGATGAGCAACGCGCAGACGTGGGCTAGTGCGGCGTTGACGGATGACGACACGTGTCTCGATGGGTTTAAAGGGGTCGAGGGTAAGGTTAAAACGGAGGTGAAGCA[G/T]TGGATGACGAAAGTGGCGAGGGTTAC[A/G]AGCAACGCGCTTTACATGATCAACCAGCTAGATGAATCACGTGGCTAGCCCCACGTAGTACGTTCTTGATGTTATGATGTGCTTGTCCTAATGGACAGTTATGATTTGGTGTTAGTTTTTTTCGTGTTTGCTTAATTGCGAGTTATCTACTATTTAAAAATGAGAGGCATTGTCCTTTTAAGTAGTTCTGATAATGGTATACTAAATAAATGGTTTATCTCTTTTTTCGGACGGTATGTCATTGTATCGTATTGTGTTGTTCCCTTCGGATTCGATAGCATGTGATTTTGTCTTGACGTGTAGTAGCGCCTTGGCTGAGCTAATGCTCTAAATAAAAGTTTTAAGTGGC
Table 14 has hereinafter been listed the Additional Information of the mark of the QTL be associated about the whole plant field resistance with to Sclerotinia, and for the exemplary forward of each polymorphic regions and the group of reverse primer sequence.
show 14:SSR and SNP mark and entirely plant for amplification and Sclerotinia (Sclerotinia) the primer sequence of the locus that the strain field resistance is associated
Figure BDA00003691347401701
Figure BDA00003691347401711
Figure BDA00003691347401721
Figure BDA00003691347401731
Figure BDA00003691347401751
Although for the purpose be aware and understand, with some details, described the present invention as described above, for a person skilled in the art, from to it is evident that reading of the present disclosure, can carry out the multiple change on form and details and not deviate from true scope of the present invention.For example, above-described whole technology, method, composition, equipment and system can be used with multiple combination.Any publication that the application quotes, patent, patent application or other file are all to be incorporated to this paper for the mode that all purpose is quoted in full, and it quotes degree just as each other publication, patent, patent application or other file individually are incorporated to this paper for the mode that all purpose is quoted in full.

Claims (39)

1. identify and show Btassica (Brassica) plant of the whole plant field resistance of the whole plant field resistance of Sclerotinia (Sclerotinia) or raising or the method for germplasm, described method is included at least one allelotrope of at least one quantitative trait locus (QTL) that in described plant or germplasm, detection is associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising, wherein said QTL is positioned to and is selected from N1, N3, N4, N7, N8, N9, N10, N11, N12, N13, N15, the linkage group of N18 or N19, wherein each described linkage group comprises at least one mark that the statistical significance with p≤0.01 is associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising, thereby identify described Brassica plants or the germplasm that will show the whole plant field resistance of the whole plant field resistance of Sclerotinia or raising.
2. method according to claim 1, wherein said QTL is positioned to and is selected between following chromosomal region:
(a) side joint comprise (i) mark CA0614 and PE0177 or (ii) interval of mark AG0093 and AG0482 on linkage group N1;
(b) side joint the interval that comprises mark CA0410 and AG0023 on linkage group N3;
(c) side joint the interval that comprises mark BG1442 and BG0106 on linkage group N4;
(d) side joint the interval that comprises mark AG0510 and CA0105 on linkage group N7;
(e) side joint the interval that comprises mark CA0837 and BG1286 on linkage group N8;
(f) side joint comprise (i) mark CA1034 and AG0441 or (ii) interval of mark AG0378 and KK66 on linkage group N9;
(g) side joint the interval that comprises mark BG0228 and PE0131 on linkage group N10;
(h) side joint comprise (i) mark CA0120 and CA0163 or (ii) interval of mark CA0120 and CA1097 on linkage group N11;
(i) side joint comprise (i) mark BG1321 and CA0991 or (ii) interval of mark CA0753 and PE0250 on linkage group N12;
(j) side joint the interval that comprises mark CA0603 and CA0736 on linkage group N13;
(k) side joint the interval that comprises mark PE0286 and AG0369 on linkage group N15;
(l) side joint comprise (i) mark BG0278 and CA0636 or (ii) interval of mark UB0315 and CA0739 on linkage group N18; With
(m) on linkage group N19 side joint and comprise (i) mark CA1107 and CA0221 or
(ii) interval of mark UB0307 and KK98G.
3. method according to claim 2, wherein said QTL is positioned to and is selected between following chromosomal region:
(a) the one or more intervals on linkage group N1, its side joint also comprises mark (i) AG0093 and PE0203 or (ii) BG0111 and BG1392 or (iii) BG1090 and AG0482 or (iv) BG1090 and PE0203 or (v) CA0614 and BG1392 or (vi) BG0988 and AG0482 or (vii) AG0243 and AG0482 or (viii) AG0243 and BG1453 or BG0988;
(b) the one or more intervals on linkage group N3, its side joint also comprises mark (i) BG1197 and AG0023 or (ii) CA0410 and BG1368 or (iii) CA0410 and BG1197;
(c) the one or more intervals on linkage group N4, its side joint also comprises mark (i) BG1442 and BG0106 or (ii) UB0181 and BG0106;
(d) the one or more intervals on linkage group N8, its side joint also comprises mark (i) BG1449 and BG1062 or (ii) CA0837 and AG0328 or (iii) CA0837 and BG1062 or (iv) CA0837 and BG1101 or (v) CA0837 and BG1286 or (vi) CA0837 and BG1449 or (vii) PE0281 and BG0647;
(e) the one or more intervals on linkage group N9, its side joint also comprises mark (i) AG0323 and BG0295 or (ii) CA1034 and AG0378 or (iii) BG1123 and AG0441;
(f) the one or more intervals on linkage group N10, its side joint also comprises mark (i) BG0228 and AG0047 or BG0255 and PE0131;
(g) the one or more intervals on linkage group N11, its side joint also comprises mark (i) BG0031 and BG1149 or (ii) BG0031 and BG1230 or (iii) BG0031 and BG1513 or (iv) CA0120 and CA0328 or (v) PE0283 and CA0163 or (vi) PE0324 and PE0283 or (vii) CA0328 and PE0324 or (viii) CA0226 and BG0713 or (ix) CA0233 and CA1080 or (x) CA0233 and AG0370;
(h) the one or more intervals on linkage group N12, its side joint also comprises mark (i) BG1321 and CA0991 or (ii) BG1321 and CA1027 or (iii) BG1321 and PE0133 or (iv) PE0063 and CA0991 or (v) PE0133 and CA0991 or (vi) CA1027 and PE0063 or (vii) CA1027 and UB0331 or (viii) CA0423 and PE0250 or (ix) AG0359 and PE0250 or (x) AG0359 and CA0896;
(i) the one or more intervals on linkage group N13, its side joint also comprises mark (i) BG0516 and AG0148 or (ii) CA0488 and AG0148 or (iii) CA0488 and CA0736 or (iv) CA0603 and AG0504 or (v) BG1288 and AG0504;
(j) the one or more intervals on linkage group N15, its side joint also comprises mark (i) CA0719 and AG0369 or (ii) PE0091 and PE0187 or (iii) PE0286 and AG0369 or (iv) PE0286 and PE0187 or (v) PE0286 and CA0719;
(k) the one or more intervals on linkage group N18, its side joint also comprises mark (i) AG0285 and CA0636 or (ii) BG0278 and CA0779 or (iii) CA0739 and CA0636 or (iv) UB0315 and CA0636 or (v) UB0315 and CA0739; With
(l) the one or more intervals on linkage group N19, its side joint also comprises mark (i) CA0552 and CA0221 or (ii) CA1107 and CA0552 or (iii) CA1107 and CA0221 or (iv) CA0221 and KK98G or (v) UB0307 and BG1241 or (vi) BG1241 and KK98G or (vii) CA0221 and BG1241.
4. according to claim 2 or method claimed in claim 3, wherein said QTL is positioned between the chromosomal region on linkage group N1, N9, N11, N12, N18 or N19.
5. method according to claim 1, wherein said mark Including Polymorphism, described polymorphism is by least one Identity of allele of described at least one quantitative trait locus (QTL) for to be associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising, and described detection comprises and identifies described polymorphism.
6. method according to claim 5, wherein said polymorphism is single nucleotide polymorphism (SNP) or simple repeated sequence (SSR).
7. method according to claim 6, wherein said detection comprises and detects at least one mark, described mark is selected from: AG0023; AG0045; AG0047; AG0070; AG0086; AG0093; AG0125; AG0148; AG0171; AG0203; AG0239; AG0243; AG0272; AG0304; AG0323; AG0324; AG0328; AG0359; AG0369; AG0370; AG0378; AG0391; AG0410; AG0441; AG0477; AG0482; AG0504; AG0510; BG0031; BG0106; BG0111; BG0119; BG0181; BG0228; BG0255; BG0278; BG0295; BG0452; BG0516; BG0647; BG0651; BG0713; BG0864; BG0869; BG0988; BG1062; BG1090; BG1101; BG1123; BG1127; BG1149; BG1182; BG1197; BG1230; BG1241; BG1244; BG1286; BG1288; BG1321; BG1368; BG1392; BG1442; BG1449; BG1453; BG1513; CA0105; CA0120; CA0163; CA0221; CA0226; CA0233; CA0328; CA0410; CA0423; CA0456; CA0488; CA0546; CA0552; CA0603; CA0614; CA0636; CA0681; CA0719; CA0736; CA0739; CA0753; CA0834; CA0837; CA0896; CA0991; CA1027; CA1032; CA1034; CA1035; CA1066; CA1080; CA1090; CA1097; CA1107; PE0012; PE0017; PE0063; PE0091; PE0131; PE0133; PE0177; PE0187; PE0203; PE0250; PE0281; PE0283; PE0286; PE0324; PE0340; PE0355; UB0015; UB0126; UB0163; UB0181; UB0196; UB0307; UB0315; UB0331; KK66; And KK98G.
8. method according to claim 7, wherein said detection comprises and detects at least one mark, described mark is selected from: AG0093; AG0304; AG0378; AG0391; AG0482; BG1149; BG1230; BG1241; BG1453; BG1513; CA0120; CA0221; CA0546; CA0739; CA1027; PE0063; PE0203; UB0163; And UB0315.
9. method according to claim 7, comprise and detect two or more marks that are arranged in two or more different linkage groups.
10. method according to claim 7, wherein said detection comprises from the genomic dna of described plant or the germplasm described mark that increases, and determines the described polymorphism be associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising that marks whether to comprise.
11. method according to claim 1, wherein said plant is swede type rape (Brassica napus); Leaf mustard (Brassica juncea); Turnip (Brassica rapa); Wild cabbage (Brassica oleracea); Or brassicacarinata (Brassica carinata).
12. method according to claim 11, wherein said plant is swede type rape (draws Kano).
13. method according to claim 12, wherein said plant is that draw the spring habit Kano.
14. method according to claim 12, wherein said plant is that draw the winter habit Kano.
15. method according to claim 12, wherein said plant is that draw the semi-winterness Kano.
16. method according to claim 1, the whole plant field resistance of wherein said whole plant field resistance or raising is to compare the disease incidence of reduction due to the allelic plant with lacking described QTL, and the allelotrope of described QTL is associated with the whole plant field resistance of described whole plant field resistance or raising.
17. method according to claim 1, the whole plant field resistance of wherein said whole plant field resistance or raising is to compare the disease severity of reduction due to the allelic plant with lacking described QTL, and the allelotrope of described QTL is associated with the whole plant field resistance of described whole plant field resistance or raising.
18. method according to claim 1, wherein said plant has the whole plant field resistance of sclerotinite (Sclerotinia sclerotiorum) or the whole plant field resistance of raising.
19. the plant by the evaluation by claim 1 or its filial generation and the second plant cross-pollination make the Sclerotinia resistance infiltrate the method in the second plant, wherein said the second plant lacks at least one allelotrope of at least one QTL detected in identified plant.
20. prepare the method for F1 crossbred seed, the F1 crossbred plant that wherein derives from described F1 crossbred seed is anti-Sclerotinia, described method comprises that wherein said the second plant lacks at least one allelotrope of at least one QTL detected in identified plant by the plant of the evaluation of claim 1 or its filial generation and the second plant cross-pollination.
21. the method for the nucleic acid that positional cloning comprises the quantitative trait locus (QTL) be associated with the whole plant field resistance of Sclerotinia whole plant field resistance or raising, described method comprises:
(a) provide the nucleic acid of the plant of self-contained mark, described mark is associated with the whole plant field resistance of Sclerotinia whole plant field resistance or raising with the statistical significance of p≤0.01, wherein said QTL is positioned to the linkage group that is selected from N1, N3, N4, N7, N8, N9, N10, N11, N12, N13, N15, N18 or N19, and wherein said linkage group comprises described mark; And
(b) nucleic acid that the clone comprises the quantitative trait locus (QTL) be associated with the whole plant field resistance of Sclerotinia whole plant field resistance or raising.
22. the method for the transgenosis dicotyledons that preparation comprises the quantitative trait locus (QTL) be associated with the whole plant field resistance of Sclerotinia whole plant field resistance or raising said method comprising the steps of:
(a) nucleic acid that will clone according to the method for claim 21 imports in the dicotyledons cell; And
(b) described cell is grown under the Growth of Cells condition.
23. identify the method for the candidate nucleic acid from dicotyledons that comprises the QTL be associated with the whole plant field resistance of Sclerotinia whole plant field resistance or raising, described method comprises:
(a) provide the nucleic acid according to the method clone of claim 21; And
(b) identify the homologue of nucleic acid described in dicotyledons.
24. the method for the quantitative trait locus (QTL) that marker assisted selection (MAS) is associated with the whole plant field resistance of whole plant field resistance to Sclerotinia or raising said method comprising the steps of:
(a) obtain the first Brassica plants, described the first Brassica plants has at least one allelotrope of the marker gene seat that the statistical significance with p≤0.01 is associated with the whole plant field resistance of the described field resistance of the whole plant to Sclerotinia or raising;
(b) by described the first Brassica plants and the hybridization of the second Brassica plants;
(c) the allelotrope assessment filial generation be associated for the whole plant field resistance with the described field resistance of the whole plant to Sclerotinia or raising; And
(d) select to have described allelic progeny plant.
25. method according to claim 24, wherein said plant is the member of segregating population.
26. method according to claim 24, wherein said marker assisted selection is used high flux screening to carry out.
27. the Brassica plants of identifying by the method for claim 24.
28. the filial generation of the Brassica plants of claim 27.
29. filial generation according to claim 28, be selected from F1, F2 and F3 filial generation.
30. nucleic acid that separate or restructuring comprises and is selected from following polynucleotide:
(a) be selected from the sequence of any one following flag sequence: AG0023 (SEQ ID NO:1); AG0045 (SEQ ID NO:2); AG0047 (SEQ ID NO:3); AG0070 (SEQ ID NO:4); AG0086 (SEQ ID NO:5); AG0093 (SEQ ID NO:6); AG0125 (SEQ ID NO:7); AG0148 (SEQ ID NO:8); AG0171 (SEQ ID NO:9); AG0203 (SEQ ID NO:10); AG0239 (SEQ ID NO:11); AG0243 (SEQ ID NO:12); AG0272 (SEQ ID NO:13); AG0304 (SEQ ID NO:14); AG0323 (SEQ ID NO:15); AG0324 (SEQ ID NO:16); AG0328 (SEQ ID NO:17); AG0359 (SEQ ID NO:18); AG0369 (SEQ ID NO:19); AG0370 (SEQ ID NO:20); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0410 (SEQ ID NO:23); AG0441 (SEQ ID NO:24); AG0477 (SEQ ID NO:25); AG0482 (SEQ ID NO:26); AG0504 (SEQ ID NO:27); AG0510 (SEQ ID NO:28); BG0031 (SEQ ID NO:29); BG0106 (SEQ ID NO:30); BG0111 (SEQ ID NO:31); BG0119 (SEQ ID NO:32); BG0181 (SEQ ID NO:33); BG0228 (SEQ ID NO:34); BG0255 (SEQ ID NO:35); BG0278 (SEQ ID NO:36); BG0295 (SEQ ID NO:37); BG0452 (SEQ ID NO:38); BG0516 (SEQ ID NO:39); BG0647 (SEQ ID NO:40); BG0651 (SEQ ID NO:41); BG0713 (SEQ ID NO:42); BG0864 (SEQ ID NO:43); BG0869 (SEQ ID NO:44); BG0988 (SEQ ID NO:45); BG1062 (SEQ ID NO:46); BG1090 (SEQ ID NO:47); BG1101 (SEQ ID NO:48); BG1123 (SEQ ID NO:49); BG1127 (SEQ ID NO:50); BG1149 (SEQ ID NO:51); BG1182 (SEQ ID NO:52); BG1197 (SEQ ID NO:53); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1244 (SEQ ID NO:56); BG1286 (SEQ ID NO:57); BG1288 (SEQ ID NO:58); BG1321 (SEQ ID NO:59); BG1368 (SEQ ID NO:60); BG1392 (SEQ ID NO:61); BG1442 (SEQ ID NO:62); BG1449 (SEQ ID NO:63); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); CA0105 (SEQ ID NO:66); CA0120 (SEQ ID NO:67); CA0163 (SEQ ID NO:68); CA0221 (SEQ ID NO:69); CA0226 (SEQ ID NO:70); CA0233 (SEQ ID NO:71); CA0328 (SEQ ID NO:72); CA0410 (SEQ ID NO:73); CA0423 (SEQ ID NO:74); CA0456 (SEQ ID NO:75); CA0488 (SEQ ID NO:76); CA0546 (SEQ ID NO:77); CA0552 (SEQ ID NO:78); CA0603 (SEQ ID NO:79); CA0614 (SEQ ID NO:80); CA0636 (SEQ ID NO:81); CA0681 (SEQ ID NO:82); CA0719 (SEQ ID NO:83); CA0736 (SEQ ID NO:84); CA0739 (SEQ ID NO:85); CA0753 (SEQ ID NO:86); CA0834 (SEQ ID NO:87); CA0837 (SEQ ID NO:88); CA0896 (SEQ ID NO:89); CA0991 (SEQ ID NO:90); CA1027 (SEQ ID NO:91); CA1032 (SEQ ID NO:92); CA1034 (SEQ ID NO:93); CA1035 (SEQ ID NO:94); CA1066 (SEQ ID NO:95); CA1080 (SEQ ID NO:96); CA1090 (SEQ ID NO:97); CA1097 (SEQ ID NO:98); Or CA1107 (SEQ ID NO:99);
(b) there is the polynucleotide sequence of at least 70% sequence identity with polynucleotide (a);
With
(c) with (a) or the polynucleotide sequence of sequence complementation (b).
31. nucleic acid separation according to claim 30 or restructuring comprises and is selected from following polynucleotide:
(a) be selected from the sequence of any one following flag sequence: AG0093 (SEQ ID NO:6); AG0304 (SEQ ID NO:14); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0482 (SEQ ID NO:26); BG1149 (SEQ ID NO:51); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); (SEQ ID NO:67); CA0221 (SEQ ID NO:69); CA0546 (SEQ ID NO:77); CA0739 (SEQ ID NO:85); Or CA1027 (SEQ ID NO:91);
(b) there is the polynucleotide sequence of at least 70% sequence identity with polynucleotide (a);
With
(c) with (a) or the polynucleotide sequence of sequence complementation (b).
32. nucleic acid separation according to claim 30 or restructuring, wherein said nucleic acid is associated with whole plant field resistance to Sclerotinia or the whole plant field resistance of raising.
33. the nucleic acid molecule separated for detection of the polymorphism be associated with the whole plant field resistance of whole plant field resistance to Sclerotinia or raising in DNA of plants, wherein said nucleic acid molecule comprises at least 15 Nucleotide, and the sequence of the continuous nucleotide of arbitrary chain equal amts of the DNA of plants be arranged in described polymorphism is identical, wherein said nucleic acid molecule comprises the sequence identical with the fragment at least 70% that is selected from following flag sequence or flag sequence: AG0023 (SEQ ID NO:1); AG0045 (SEQ ID NO:2); AG0047 (SEQ ID NO:3); AG0070 (SEQ ID NO:4); AG0086 (SEQ ID NO:5); AG0093 (SEQ ID NO:6); AG0125 (SEQ ID NO:7); AG0148 (SEQ ID NO:8); AG0171 (SEQ ID NO:9); AG0203 (SEQ ID NO:10); AG0239 (SEQ ID NO:11); AG0243 (SEQ ID NO:12); AG0272 (SEQ ID NO:13); AG0304 (SEQ ID NO:14); AG0323 (SEQ ID NO:15); AG0324 (SEQ ID NO:16); AG0328 (SEQ ID NO:17); AG0359 (SEQ ID NO:18); AG0369 (SEQ ID NO:19); AG0370 (SEQ ID NO:20); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0410 (SEQ ID NO:23); AG0441 (SEQ ID NO:24); AG0477 (SEQ ID NO:25); AG0482 (SEQ ID NO:26); AG0504 (SEQ ID NO:27); AG0510 (SEQ ID NO:28); BG0031 (SEQ ID NO:29); BG0106 (SEQ ID NO:30); BG0111 (SEQ ID NO:31); BG0119 (SEQ ID NO:32); BG0181 (SEQ ID NO:33); BG0228 (SEQ ID NO:34); BG0255 (SEQ ID NO:35); BG0278 (SEQ ID NO:36); BG0295 (SEQ ID NO:37); BG0452 (SEQ ID NO:38); BG0516 (SEQ ID NO:39); BG0647 (SEQ ID NO:40); BG0651 (SEQ ID NO:41); BG0713 (SEQ ID NO:42); BG0864 (SEQ ID NO:43); BG0869 (SEQ ID NO:44); BG0988 (SEQ ID NO:45); BG1062 (SEQ ID NO:46); BG1090 (SEQ ID NO:47); BG1101 (SEQ ID NO:48); BG1123 (SEQ ID NO:49); BG1127 (SEQ ID NO:50); BG1149 (SEQ ID NO:51); BG1182 (SEQ ID NO:52); BG1197 (SEQ ID NO:53); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1244 (SEQ ID NO:56); BG1286 (SEQ ID NO:57); BG1288 (SEQ ID NO:58); BG1321 (SEQ ID NO:59); BG1368 (SEQ ID NO:60); BG1392 (SEQ ID NO:61); BG1442 (SEQ ID NO:62); BG1449 (SEQ ID NO:63); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); CA0105 (SEQ ID NO:66); CA0120 (SEQ ID NO:67); CA0163 (SEQ ID NO:68); CA0221 (SEQ ID NO:69); CA0226 (SEQ ID NO:70); CA0233 (SEQ ID NO:71); CA0328 (SEQ ID NO:72); CA0410 (SEQ ID NO:73); CA0423 (SEQ ID NO:74); CA0456 (SEQ ID NO:75); CA0488 (SEQ ID NO:76); CA0546 (SEQ ID NO:77); CA0552 (SEQ ID NO:78); CA0603 (SEQ ID NO:79); CA0614 (SEQ ID NO:80); CA0636 (SEQ ID NO:81); CA0681 (SEQ ID NO:82); CA0719 (SEQ ID NO:83); CA0736 (SEQ ID NO:84); CA0739 (SEQ ID NO:85); CA0753 (SEQ ID NO:86); CA0834 (SEQ ID NO:87); CA0837 (SEQ ID NO:88); CA0896 (SEQ ID NO:89); CA0991 (SEQ ID NO:90); CA1027 (SEQ ID NO:91); CA1032 (SEQ ID NO:92); CA1034 (SEQ ID NO:93); CA1035 (SEQ ID NO:94); CA1066 (SEQ ID NO:95); CA1080 (SEQ ID NO:96); CA1090 (SEQ ID NO:97); CA1097 (SEQ ID NO:98); Or CA1107 (SEQ ID NO:99).
34. the nucleic acid molecule of separation according to claim 33, be selected from any one in SEQ ID NO:126-323.
35. the nucleic acid molecule of separation according to claim 33, wherein said flag sequence is selected from any one in following: AG0093 (SEQ ID NO:6); AG0304 (SEQ ID NO:14); AG0378 (SEQ ID NO:21); AG0391 (SEQ ID NO:22); AG0482 (SEQ ID NO:26); BG1149 (SEQ ID NO:51); BG1230 (SEQ ID NO:54); BG1241 (SEQ ID NO:55); BG1453 (SEQ ID NO:64); BG1513 (SEQ ID NO:65); (SEQ ID NO:67); CA0221 (SEQ ID NO:69); CA0546 (SEQ ID NO:77); CA0739 (SEQ ID NO:85); Or CA1027 (SEQ ID NO:91).
36. the nucleic acid molecule of separation according to claim 35, be selected from any one in SEQ ID NO:136,137,152,153,166,167,168,169,176,177,226,227,232,233,234,235,252,253,254,255,258,259,262,263,278,279,294,295,306 or 307.
37. the test kit of the QTL be associated for the whole plant field resistance for to Sclerotinia screening plant or germplasm, comprise container, comprises in described container:
(a) nucleic acid molecule of the separation of one or more claims 33; With
(b) specification sheets of the QTL screening plant be associated for the whole plant field resistance of the whole plant field resistance for to Sclerotinia or raising.
38., according to the described test kit of claim 38, comprise at least one for the component for the described plant of described QTL high flux screening or germplasm.
39. show the Brassica plants to the whole plant field resistance of the whole plant field resistance of Sclerotinia or raising, described plant includes the allelotrope that is beneficial to Sclerotinia whole plant field resistance at least one QTL, and described QTL is positioned to and is selected between following chromosomal region:
(a) side joint comprise (i) mark CA0614 and PE0177 or (ii) interval of mark AG0093 and AG0482 on linkage group N1;
(b) side joint the interval that comprises mark CA0410 and AG0023 on linkage group N3;
(c) side joint the interval that comprises mark BG1442 and BG0106 on linkage group N4;
(d) side joint the interval that comprises mark AG0510 and CA0105 on linkage group N7;
(e) side joint the interval that comprises mark CA0837 and BG1286 on linkage group N8;
(f) side joint comprise (i) mark CA1034 and AG0441 or (ii) interval of mark AG0378 and KK66 on linkage group N9;
(g) side joint the interval that comprises mark BG0228 and PE0131 on linkage group N10;
(h) side joint comprise (i) mark CA0120 and CA0163 or (ii) interval of mark CA0120 and CA1097 on linkage group N11;
(i) side joint comprise (i) mark BG1321 and CA0991 or (ii) interval of mark CA0753 and PE0250 on linkage group N12;
(j) side joint the interval that comprises mark CA0603 and CA0736 on linkage group N13;
(k) side joint the interval that comprises mark PE0286 and AG0369 on linkage group N15;
(l) side joint comprise (i) mark BG0278 and CA0636 or (ii) interval of mark UB0315 and CA0739 on linkage group N18; With
(m) side joint comprise (i) mark CA1107 and CA0221 or (ii) interval of mark UB0307 and KK98G on linkage group N19.
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